Immunogenic arginase peptides

ABSTRACT

The present invention relates to immunogenic polypeptide fragments of a human Arginase protein. The fragments are in particular useful for the treatment or prevention of cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase application under 35 U.S.C. § 371of PCT International Application No. PCT/EP2017/075443, filed Oct. 6,2017, which claims priority to European Application No. 16192794.2,filed Oct. 7, 2016, each of which are hereby incorporated by referencein its entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 28,703 Byte ASCII (Text) file named“37700-US-1-PCT.TXT,” created on Jun. 28, 2019.

TECHNICAL FIELD

The present invention relates to novel peptide compounds, such asfragments of Arginase 1, as well as compositions, uses, and kit-of-partscomprising these peptide compounds. Furthermore, the invention concernsnucleic acids, vectors, and host cells expressing said peptidecompounds, for use in a method for treatment or prevention of a cancer,either alone or when administered simultaneously or sequentially with anadditional cancer therapy.

BACKGROUND ART

Arginase is an enzyme that catalyses a reaction which converts the aminoacid L-arginine into L-omithine and urea. This depletes themicroenvironment of arginine and leads to a suppression oftumor-specific cytotoxic T-cell responses. Increased Arginase activityhas been detected in the cancer cells of patients with breast, lung,colon or prostate cancer [1]. It has been shown both in vitro and invivo that mouse macrophages transfected with a rat Arginase gen promotethe proliferation of co-cultured tumour cells [2]. Furthermore inductionof Arginase expression by macrophages has been shown to increase tumourvascularization through polyamine synthesis. The results of a murinelung carcinoma model showed that there existed a subpopulation of maturetumor-associated myeloid cells that expressed high levels of Arginase.These tumor-associated myeloid cells depleted the extracellularL-Arginine which inhibited antigen-specific proliferation of the tumorinfiltrating lymphocytes (TILs). Injection of an Arginase inhibitorblocked the growth of the lung carcinoma in the mice. This shows howinduction of Arginase expression in tumor cells and tumor associatedmyeoloid cells might promote tumor growth by suppression of theanti-tumor immune responses through negative effects on TILs.

MDSCs (myeloid-derived suppressor cells) inhibit the activation,proliferation, and cytotoxicity of effector T cells and natural killercells, as well as induce Treg differentiation and expansion. Both cancercells and MDSCs can suppress T cells by manipulating L-argininemetabolism via the enzymes nitric-oxide synthase (NOS) and arginase.Many tumours exhibit increased expressions of arginase and inducible NOS(iNOS), leading to arginine depletion from the tumour microenvironment[1]. Several studies emphasize the importance of this altered tumourarginine metabolism in the suppression of tumour-specific T-cellresponses, and it was recently demonstrated that AML blasts show anarginase-dependent ability to inhibit T-cell proliferation andhematopoietic stem cells. Furthermore, arginase and iNOS inhibitorsreduce the suppressive activity of AML [2].

SUMMARY OF THE INVENTION

The present inventors have identified new immunogenic epitopes fromArginase 1. Furthermore, peripheral blood mononuclear cells (PBMC) frommelanoma patients have been analysed for the presence of specific T-cellresponses against Arginase 1-derived peptides and strong immuneresponses against the new immunogenic epitopes were detected. Moreover,frequent immune responses were detected against several peptidefragments. It has also been shown that the immune responses towardsArginase 1 indeed were mediated by CD4 and CD8 T cells, and that bothCD8 and CD4 T cells can recognize Arginase 1 derived peptides on thesurface of target cells.

The development of novel immune therapies for cancer requires a thoroughunderstanding of the molecules that are involved in the pathogenesis aswell as the specific proteins recognized by the immune system. In theclinical setting the induction of Arginase specific immune responsescould in addition to the killing of cancer cells support anti-cancerimmune responses in general by suppressing the immune suppressivefunction of Arginase expressing cells especially MDSC andtumor-associated macrophages (TAMs). Hence, since Arginase-expressingcells antagonize the desired effects of other immunotherapeuticapproaches targeting myeloid dendritic cells e.g. by vaccination, wouldconsequently be highly synergistic with additional anti-cancerimmunotherapy.

Provided herein is an isolated, immunogenic polypeptide fragment of ahuman Arginase protein of SEQ ID NO: 1 (Arginase 1) or SEQ ID NO: 60(Arginase 2). The fragment is typically up to 8, 9, 10, 15, 20, 25, 30,45, 50 or 55 amino acids in length. The fragment may comprise orconsists of a sequence of at least 8, 9, 10, 20, 30, 40 or 50consecutive amino acids of either of SEQ ID NO: 52 or SEQ ID NO: 58. Thepolypeptide fragment may comprise or consist of the amino acid sequenceof any one of SEQ ID NOs: 52, 50, 51, 34, 35, 36, 37, 9, 53, 54, 2 to33, or 38 to 49.

The polypeptide fragment may have one or more of the followingadditional features:

-   -   a. the C terminal amino acid is replaced with the corresponding        amide; and/or    -   b. the L at the position corresponding to position 190 of SEQ ID        NO: 1 is replaced with I; and/or    -   c. the R at the position corresponding to position 205 of SEQ ID        NO: 1 is replaced with K; and/or    -   d. at least one additional moiety is attached to the N and/or C        terminus, optionally wherein said additional moiety is a        hydrophilic amino acid such as R or K; and/or    -   e. lacks or has reduced arginase activity relative to the        corresponding full length arginase.        Also provided is a composition comprising a said fragment, a        pharmaceutically acceptable diluent or carrier, and optionally        an adjuvant. Said composition or said fragment may be for use in        a method of treating or preventing a disease or condition such        as cancer, or for use in the manufacture of a medicament for        treating or preventing a disease or condition such as cancer.        Said composition may optionally be described as a vaccine. Also        provided is a method of treating or preventing a disease or        condition such as cancer, the method comprising administering        said composition or said fragment to a subject in need thereof.

The polypeptide fragment may be interchangeably described herein as apeptide compound or a peptide.

In one aspect the present disclosure concerns a peptide compound ofArginase 1 selected from:

a) a peptide fragment of SEQ ID NO 1 consisting of a consecutivesequence of from 8 to 321 amino acids,

b) a functional homologue having at least 70%, 80%, 90%, or 95% identityto SEQ ID NO 1 or the peptide fragment of a), and

c) a functional analogue wherein at least one amino acid has beendeleted, inserted and/or substituted in SEQ ID NO 1 or the peptidefragment of a),

and wherein the C-terminal amino acid of any one of a), b) or c) alsocomprises the amide; or a pharmaceutically acceptable salt thereof. Inthe above-mentioned context, “functional” means “capable of stimulatingan immune response to the Arginase of SEQ ID NO: 1”. The homologue b)and analogue c) preferably have reduced (or zero) arginase function.

In an embodiment the peptide compound is selected from a) a peptidefragment of SEQ ID NO 1 consisting of a consecutive sequence of from 8to 321 amino acids, wherein the C-terminal amino acid also comprises theamide; or a pharmaceutically acceptable salt thereof. In a furtherembodiment the peptide fragment consists of a consecutive sequence inthe range of from 8 to 300 amino acids, 8 to 250 amino acids, 8 to 200amino acids, 8 to 150 amino acids, 8 to 120 amino acids, e.g. 10 to 100amino acids, 20 to 80 ami-no acids, 30 to 60 amino acids, 40 to 50 aminoacids. In a still further embodiment the peptide fragment of SEQ ID NO 1is selected from the group consisting of ARG(1-310), ARG(1-301),ARG(1-291), ARG(11-322), ARG(21-322), ARG(30-322), ARG(40-322),ARG(11-310), ARG(11-301), ARG(11-291), ARG(21-310), ARG(21-301),ARG(21-291), ARG(30-310), ARG(30-301), ARG(30-291), ARG(40-310),ARG(40-301), and ARG(40-291).

In a further embodiment the arginase activity is reduced compared toArginase 1 as measured by an arginase activity assay. In one embodimentthe arginase activity is reduced to inactivity. In another embodimentthe arginase activity assay is selected from the Arginase ActivityColorimetric Assay Kit (BioVision Arginase assay # K755-100).

In a still further embodiment the consecutive sequence comprises one ormore sequences selected from any one of SEQ ID NOs: 52, 50, 51, 37, 36,35, 34, 9, 53, 54, 2 to 33, or 38 to 39. In one embodiment theconsecutive sequence comprises the sequence selected from SEQ ID NO 52,50, 51, 37, 36, 35, 34, 9, 53 or 54.

In a further embodiment the peptide fragment under a), the functionalhomologue under b), or the functional analogue under c) is functional inthe sense that it activates T cells that recognizes Arginase 1expressing cells. In one embodiment the activation is determined by theELISPOT assay described herein.

Also disclosed herein is a nucleic acid encoding the peptide compound ofthe present invention. The peptide compound of the present invention isselected from any one of the above embodiments. In one embodiment thenucleic acid is selected from the group consisting of DNA and RNA.

Also disclosed herein is a vector comprising the nucleic acid of thepresent invention. The nucleic acid of the present invention is selectedfrom any one of the above embodiments, and the peptide compound of thepresent invention is selected from any one of the above embodiments. Inone embodiment the vector is selected from a virus vector.

In a further aspect the present disclosure relates to a host cellcomprising the vector of the present invention. The vector is selectedfrom any one of the above embodiments, the nucleic acid is selected fromany one of the above embodiments, and the peptide compound is selectedfrom any one of the above embodiments. In one embodiment the host cellis selected from a mammalian cell.

The vector preferably comprises a nucleic acid encoding an inactivesequence of Arginase 1, that is a sequence which lacks arginasefunction.

Any peptide, nucleic acid, vector, or host cell described herein may beprovided in a composition, optionally together with a pharmaceuticallyacceptable additive, such as a carrier or diluent. The composition mayoptionally also comprise an adjuvant. The composition may be for use asa medicament. The composition may be for use in the manufacture of amedicament for treating or preventing a disease.

The composition may be for use in a method for treatment or preventionof a disease, disorder or condition selected from cancer. In oneembodiment the cancer is a tumor forming cancer disease. The adjuvantmay be selected from the group consisting of bacterial DNA basedadjuvants, oil/surfactant based adjuvants, viral dsRNA based adjuvants,imidazochinilines, and a Montanide ISA adjuvant.

A method of treatment or prevention of a disease as disclosed herein maycomprise administering to a subject an effective amount of:

a) a composition as described above, and

b) a composition comprising at least one second active ingredient,selected from an immunostimulating compound, such as an interleukin,e.g. IL-2 and or IL-21, an anti-cancer agent, such as a chemotherapeuticagent, e.g. Actimide, Azacitidine, Azathio-prine, Bleomycin,Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophospha-mide,Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin,Epirubicin, Etoposide, Fludarabine, Fluorouracil, Gemcitabine,Hydroxyurea, Idarubicin, Irinotec-an, Lenalidomide, Leucovorin,Mechlorethamine, Melphalan, Mercaptopurine, Metho-trexate, Mitoxantrone,Oxaliplatin, Paclitaxel, Pemetrexed, Revlimid, Temozolomide, Teniposide,Thioguanine, Valrubicin, Vinblastine, Vincristine, Vindesine andVinorelbine, or a checkpoint inhibitor, e.g. antibody such as nivolumabor pembrolizumab.

In The provided compositions may be administered simultaneously orsequentially. The compositions may be provided as components in akit-of-parts.

In a further aspect the present disclosure relates to a method oftreating a clinical condition characterized by expression of Arginase 1of SEQ ID NO 1, the method comprising administering to an individualsuffering from said clinical condition an effective amount of thepeptide, the nucleic acid, the vector or the host cell described above.

In a further aspect the present disclosure relates to a method ofstimulation of arginase 1 specific T-cells, such as CD4 and CD8 T-cells,in a cancer patient, the method comprising administering to the cancerpatient an effective amount of the peptide compound, or the nucleicacid, or the vector, or the host cell as described above.

In a still further aspect the present disclosure relates to a method ofsuppressing an immune suppressive function of Arginase 1 expressingcells, in a cancer patient, the method comprising administering to thecancer patient an effective amount of the peptide compound, or thenucleic acid, or the vector, or the host cell as described above.

In a further aspect the present disclosure relates to use of the peptidecompound, or the nucleic acid, or the vector, or the host cell, asdescribed above for the manufacture of a medicament, such as animmunotherapeutic composition or vaccine, for the treatment orprevention of a cancer, wherein said cancer is optionally characterizedby expression of Arginase 1.

In a still further aspect the present disclosure relates to the peptidecompound, or the nucleic acid, or the vector, or the host cell, for usein a method for treatment or prevention of a cancer, optionally whenadministered simultaneously or sequentially with an additional cancertherapy.

The additional cancer therapy may be selected from the group consistingof a cytokine therapy, a T-cell therapy, an NK therapy, an immune systemcheckpoint inhibitor, chemotherapy, radiotherapy, immunostimulatingsubstances, gene therapy, antibodies and dendritic cells. In oneembodiment the additional cancer therapy is selected from an immunesystem checkpoint inhibitor, such as a checkpoint blocking antibody(e.g. selected from nivolumab or pemrbolizumab), or is selected from thegroup consisting of Actimide, Azacitidine, Azathioprine, Bleomycin,Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide,Cytarabine, Dauno-rubicin, Docetaxel, Doxifluridine, Doxorubicin,Epirubicin, Etoposide, Fludarabine, Fluor-ouracil, Gemcitabine,Hydroxyurea, Idarubicin, Irinotecan, Lenalidomide, Leucovorin,Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitoxantrone,Oxaliplatin, Paclitaxel, Pemetrexed, Revlimid, Temozolomide, Teniposide,Thioguanine, Valrubicin, Vinblastine, Vincristine, Vindesine andVinorelbine.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows T cell responses in samples from 6 cancer patients to thepeptides consisting of each of the amino acids sequences of SEQ ID NOs:2 to 16 (Arg1 to Arg15).

FIG. 2 shows T cell responses in samples from 6 cancer patients againstthe peptide consisting of each of the amino acid sequence of SEQ ID NO:9 (Arg8).

FIG. 3 shows T cell responses in samples from 3 cancer patients to thepeptides of consisting of each of the amino acid sequences of SEQ IDNOs: 18 to 48 (Arg1-1 to Arg1-31).

FIG. 4 shows T cell responses in samples from 8 healthy individualsagainst the peptides consisting of the amino acid sequence of each ofSEQ ID NO: 9 (Arg8), SEQ ID NO: 21 (Arg1-4), SEQ ID NO: 29 (Arg1-12),SEQ ID NO: 34 (Arg1-17), SEQ ID NO: 35 (Arg1-18), SEQ ID NO: 36(Arg1-19), SEQ ID NO: 37 (Arg1-20), SEQ ID NO: 40 (Arg1-23), SEQ ID NO:44 (Arg1-17).

FIG. 5 shows CD8 positive culture can kill target cells loaded with thepeptide consisting of each of the amino acid sequence of SEQ ID NO: 9(Arg8).

FIG. 6 shows killing of Arginase positive cancer (melanoma) cell linesFM3 and FM93 by Arginase specific T cells.

FIG. 7 shows flow cytometric analysis of CD4 Tcells from a cancerpatient, assessed by intracellular staining for IFN-g (PE-Cy7A) andTNF-alpha (APC-A) following culture either without (top panel) or with(bottom panel) stimulation with the peptide consisting of each of theamino acid sequence of SEQ ID NO: 9 (Arg8). Similar results are shown inFIG. 8.

FIG. 9 shows that multiple arginase-1 peptides are recognized by PBMCsfrom 3 melanoma patients (9A) and 8 healthy donors (9B) when assessed byIFNγ ELISPOT. Peptides are described by reference to the start and endpositions of their sequences within the sequence of human Arginase 1.Spot counts are given as a difference between averages of the wellsstimulated with the peptide and control wells. Peptide and controlstimulations were performed in duplicates or triplicates.

FIG. 10 shows that the region corresponding to positions 161 to 210 ofSEQ ID NO: 1 is widely recognized by cancer patient and healthy donorPBMCs

A—shows the proportion of CD4+ T cells in tumour infiltratinglymphocytes (TILs) from melanoma patient which release IFNγ in responseto the peptides consisting of the amino acid sequences of each of SEQ IDNO: 34 (Arg161-180), SEQ ID NO: 35 (Arg171-190) and SEQ ID NO: 36(Arg181-200). Peptide names refer to the start and end positions oftheir sequences within the sequence of human Arginase 1B—Left: Responses against Arg(161-190) peptide in PBMCs from 5 selectedcancer patients and four healthy donors. Right: Exemplary ELISPOT wellsfor responses against Arg(161-190) peptide or control in 2 healthydonors (HD.) and 2 cancer patients (MM.).C—Left: Responses against Arg(181-210) peptide in PBMCs from 5 selectedcancer patients and four healthy donors. Right: Exemplary ELISPOT wellsfor responses against Arg(161-190) peptide in 2 healthy donors (HD.) and2 cancer patients (MM.).Spot counts are given as a difference between averages of the wellsstimulated with the peptide and control wells. Peptide and controlstimulations were performed in triplicates.

FIG. 11 shows further analysis of responses to the region correspondingto positions 161 to 210 of SEQ ID NO: 1

A—Top shows exemplary flow cytometry analysis of CD4 Tcells from acancer patient, assessed by intracellular staining for IFN-g (PE-Cy7A)and TNF-alpha (APC-A) release following 8 hour stimulation with thepeptide Arg(161-190) or unstimulated without peptide. Bottom shows theproportion TNF-a releasing CD4+ T cells in this assay for 2 healthydonors (HD.) and 1 cancer patient (MM.).B—summary of IFNγ ELISPOT results following stimulation of PBMCs from 3cancer patients with the peptide Arg(161-190) alone, or with antibodyblocking of expression of HLA Class I (anti-Class I; W6/32) or HLA ClassII (anti-Class II; Tü39). Spot counts are given as a difference betweenthe wells stimulated with the peptide and control wells. Peptide andcontrol stimulations were performed in duplicates or triplicates.C—Left: IFNγ ELISPOT responses from Arginase specific CD4 T cells(produced by repeated stimulation with the peptide consisting of theamino acid sequence of SEQ ID NO: 9 (ArgShort)) when stimulated withArgShort, the 30mer consisting of the sequence from positions 161 to 190of Arginase 1 (Arg 161-190), or the 50mer consisting of the sequencefrom positions 161 to 210 of Arginase 1 (Arg 161-210), or whenunstimulated (control). Right: Exemplary flow cytometric analysis of theArginase specific CD4 T cells assessed by intracellular staining forIFN-g (PE-Cy7A) and TNF-alpha (APC-A) release following 8 hourstimulation with the peptide Arg(161-190) or control.

FIG. 12 shows Arginase-specific T cells recognize arginase-expressingimmune cells A and C—IFNγ response by the arginase specific T cellcultures from two different melanoma patients (MM01 and MM05) toautologous dendritic cells electroporated and transfected withirrelevant control mRNA (DC Mock) or Arginase-1 mRNA (DC Arg mRNA).Effector to target ratio 10:1.

B and D—IFNγ response by the arginase specific T cell cultures from twodifferent melanoma patients to autologous B cells electroporated andtransfected with irrelevant control mRNA (DC Mock) or Arginase-1 mRNA(DC Arg mRNA) Effector to target 2:1.

E—Bottom: IFNγ response by arginase specific T cell culture towardsautologous dendritic cells electroporated and transfected withirrelevant control mRNA (DC Mock), arginase mRNA (DC Arg mRNA) orarginase mRNA containing DC-LAMP signal sequence (DC LAMP Arg mRNA).Top: representative ELISPOT well images. Control and transfected cellstimulations were performed in duplicates or triplicates.

FIG. 13 is a sequence alignment of human Arginase 1 (SEQ ID NO: 1) andmurine Arginase 1 (SEQ ID NO: 59).

FIG. 14 shows that arginase specific immune responses are increased inC57BL/6 mice following vaccination with different peptides disclosedherein.

FIG. 15 shows that arginase specific immune responses are increased inBalb/c mice following vaccination with different peptides disclosedherein.

FIG. 16 shows reduction in tumour volumes in a mouse model of cancervaccination with different peptides disclosed herein.

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO: 1 is the wild type amino acid sequence of human Arginase 1

SEQ ID NOS: 2 to 54 are the amino acid sequences of peptide fragments ofhuman Arginase 1

SEQ ID NOs: 55 to 57 are the amino acid sequences of peptide fragmentsof murine Arginase 1

SEQ ID NOs: 58 is the amino acid sequence of a peptide fragment of humanArginase 2

SEQ ID NO: 59 is the wild type amino acid sequence of murine Arginase 1

SEQ ID NO: 60 is the wild type amino acid sequence of human Arginase 2

SEQ ID NO: 61 is an alternative sequence of human Arginase 1

DETAILED DESCRIPTION OF THE INVENTION

The present invention is concerned with immunogenic polypeptidefragments of Arginase proteins. Such fragments may be useful asvaccines. By “immunogenic” it is meant that a polypeptide fragment iscapable of eliciting an immune response, preferably a T-cell response,in at least one individual after administration to said individual. Apolypeptide may be identified as immunogenic using any suitable method,including in vitro methods. For example, a peptide may be identified asimmunogenic if it has at least one of the following characteristics:

-   -   (i) It is capable of eliciting IFN-γ-producing cells in a PBL        population of at least one cancer patient as determined by an        ELISPOT assay, and/or    -   (ii) It is capable of in situ detection in a sample of tumor        tissue of CTLs that are reactive with the corresponding        arginase; and/or    -   (iii) It is capable of inducing the in vitro growth of specific        T-cells.        Methods suitable for determining whether a polypeptide is        immunogenic active are also provided in the Examples section        below.

The inventors have determined that multiple regions of the Arginase 1sequence of SEQ ID NO: 1 are immunogenic. These include SEQ ID NOs: 52,50, 51, 37, 36, 35, 34, 9, 53, 54, 2 to 33, or 38 to 49. SEQ ID NO: 52corresponds to the region of Arginasel from position 161 to 210 of SEQID NO: 1. This region may be described as a hotspot for immunogenicitybecause it comprises sequences against which T cell responses aredetected most frequently amongst cancer patients and healthy subjects.The immunogenic polypeptide may be upto 50 or 55 amino acids in lengthand/or comprise at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or all 50consecutive amino acids of SEQ ID NO: 52. Said consecutive amino acidsmay preferably comprise or consist of any one of SEQ ID NOs: 50, 51, 34,35, 36, 37, 9, 53 and 54. Each of SEQ ID NOs: 50, 51, 34, 35, 36, 37, 9,53 and 54 comprises sequences from within the sequence of SEQ ID NO: 52.Specifically, each of SEQ ID NOs: 50, 51, 34, 35, 36, 37, 9, 53 and 54corresponds to the regions of SEQ ID NO: 1 defined by positions 161-190,181-210, 161-180, 171-190, 181-200, 191-210, 174-182, 172-179 and193-200, respectively.

The immunogenic polypeptide fragment may be upto 30, 35, 40, 45, 50 or55 amino acids in length and comprise at least 8, 9, 10, 15, 20, 25 orall 30 consecutive amino acids of SEQ ID NO: 50 or 51. The immunogenicpolypeptide fragment may be upto 20, 25, 30, 35, 40, 45, 50 or 55 aminoacids in length and comprise at least 8, 9, 10, 15 or all 20 consecutiveamino acids of any one of SEQ ID NOs: 37, 36, 35, 34.

The polypeptide fragment preferably comprises at least 8, 9, 10, 15 orall 20 consecutive amino acids from the region defined by positions181-200 of SEQ ID NO: 1 (that is SEQ ID NO: 36) or most preferably atleast 8, 9, 10, 15 or all 20 consecutive amino acids from the regiondefined by positions 191-200 of SEQ ID NO: 1 (that is SEQ ID NO: 37).The polypeptide fragment may comprise at least 8, 9, 10, 15 or all 20consecutive amino acids from the region defined by positions 161-180 ofSEQ ID NO: 1 (that is SEQ ID NO: 34) or positions 171-190 of SEQ ID NO:1 (that is SEQ ID NO: 35). In the former (SEQ ID NO: 34), the cysteinecorresponding to position 168 may optionally be replaced by conservativesubstitution, e.g. to improve solubility or stability.

The immunogenic polypeptide fragment may be upto 8 or 9 amino acids inlength and may comprise at least 8 or 9 consecutive amino acids of anyone of SEQ ID NOs: 9, 53 or 54. The immunogenic polypeptide fragment maycomprise or consist of the amino acid sequence of any one of SEQ ID NOs:52, 50, 51, 34, 35, 36, 37, 9, 53, 54, 2 to 33, or 38 to 49.

The inventors also identified responses in sequences which correspond toother regions of SEQ ID NO: 1, including the regions defined by (or atleast partially overlapping with) positions 221-240 (see SEQ ID NOs: 40,7, 14); 271-290 (see SEQ ID NOs: 45, 8, 10, 11, 12); 111-130 (see SEQ IDNOs: 29); 1-20 (see SEQ ID NOs: 18); 61-80 (see SEQ ID NOs: 24); 131-150(see SEQ ID NOs: 31, 4); 141-160 (see SEQ ID NOs: 32, 4); 51-70 (see SEQID NOs: 23); 151-170 (see SEQ ID NOs: 33, 4); 211-240 (see SEQ ID NOs:39, 6); and 281-300 (see SEQ ID NOs: 46). The immunogenic polypeptidefragment may thus comprise at least 8, 9 or more consecutive amino acidsfrom any one of these regions or sequences. The immunogenic polypeptidefragment may comprise or consist of the amino acid sequence of any oneof these regions or sequences.

Human Arginase 2 is related to Arginase 1 and there are somesimilarities in sequence (see SEQ ID NO: 60 versus SEQ ID NO: 1,respectively). The regions of interest in Arginase 1 are thus alsolikely to be of interest in Arginase 2. Accordingly, any one of thesequences described in the preceding section may have any one or moreamino acids replaced with the one or more amino acids in thecorresponding positions in Arginase 2. The entirety of the said sequencemay be replaced with the corresponding sequence in Arginase 2. Theimmunogenic polypeptide may comprise or consist of a sequence of SEQ IDNO: 60 which is defined by the positions in SEQ ID NO: 60 thatcorrespond to the positions in SEQ ID NO: 1 of any one of SEQ ID NOs:52, 50, 51, 34, 35, 36, 37, 9, 53, 54, 2 to 33, or 38 to 49. Forexample, the hotspot region defined by positions 161-210 of SEQ ID NO: 1corresponds to positions 180-229 of SEQ ID NO: 60 which are representedherein as SEQ ID NO: 58. Thus, references to SEQ ID NO: 52 may bereplaced by references to SEQ ID NO: 58. The immunogenic polypeptide maycomprise at least 8, 9, or preferably at least 20 or 30 consecutiveamino acids of SEQ ID NO: 58.

Human Arginase 1 is also highly similar to murine Arginase 1 (see SEQ IDNO: 59 versus SEQ ID NO: 1, respectively, plus FIG. 13). Accordingly,any one of the sequences described in the preceding section for humanArginase 1 may have any one or more amino acids replaced with the one ormore amino acids in the corresponding positions in murine Arginase 1.The entirety of the said sequence may be replaced with the correspondingsequence in murine Arginase 1. The immunogenic polypeptide may compriseor consist of a sequence of SEQ ID NO: 59 which is defined by thepositions in SEQ ID NO: 59 that correspond to the positions in SEQ IDNO: 1 of any one of SEQ ID NOs: 52, 50, 51, 34, 35, 36, 37, 9, 53, 54, 2to 33, or 38 to 49. For example, the hotspot region defined by positions161-210 of SEQ ID NO: 1 corresponds to positions 161-210 of SEQ ID NO:59, which are represented herein as SEQ ID NO: 57. Thus, references toSEQ ID NO: 52 may be replaced by references to SEQ ID NO: 57. Theimmunogenic polypeptide may comprise at least 8, 9, or preferably atleast 20 or 30 consecutive amino acids of SEQ ID NO: 57. In effect, thismeans that the L at the position corresponding to position 190 of SEQ IDNO: 1 may be replaced with I and/or that the R at the positioncorresponding to position 205 of SEQ ID NO: 1 is replaced with K, in anypolypeptide fragment described herein which encompasses those residues.

In any polypeptide fragment described herein, the C terminal amino acidmay optionally be replaced with the corresponding amide, to improvesolubility and/or to aid with manufacture/isolation. Similarly, thepolypeptide may have attached at the N and/or C terminus at least oneadditional moiety to improve solubility and/or to aid withmanufacture/isolation. Suitable moieties include hydrophilic aminoacids. For example, the amino acids KR may be added at the N terminusand/or the amino acids RK may be added in order at the C terminus.

Any polypeptide fragment described herein preferably has reducedarginase activity relative to the corresponding full-length arginase. Areduction in arginase activity may include the reduction to inactivity.A suitable assay for arginase activity is the Arginase ActivityColorimetric Assay Kit (BioVision Arginase assay # K755-100).

In one aspect the present disclosure concerns a peptide compound ofArginase 1 selected from:

a) a peptide fragment of SEQ ID NO 1 consisting of a consecutivesequence of from 8 to 321 amino acids,

b) a functional homologue having at least 70%, 80%, 90%, or 95% identityto SEQ ID NO 1 or the peptide fragment of a), and

c) a functional analogue wherein at least one amino acid has beendeleted, inserted and/or substituted in SEQ ID NO 1 or the peptidefragment of a),

and wherein the C-terminal amino acid of any one of a), b) or c) alsocomprises the amide; or a pharmaceutically acceptable salt thereof.

In an embodiment the peptide compound is selected from b) a functionalhomologue having at least 70%, 80%, 90%, or 95% identity to SEQ ID NO 1or the peptide fragment of a), wherein the C-terminal amino acid alsocomprises the amide; or a pharmaceutically acceptable salt thereof. Inone embodiment the functional homologue has at least 80% identity to SEQID NO 1. In a further embodiment the functional homologue has at least90% identity to SEQ ID NO 1. In a further embodiment the functionalhomologue has at least 95% identity to SEQ ID NO 1. In a furtherembodiment the functional homologue has at least 70% identity to thepeptide fragment of a). In a further embodiment the functional homologuehas at least 80% identity to the peptide fragment of a). In a furtherembodiment the functional homologue has at least 90% identity to thepeptide fragment of a). In a further embodiment the functional homologuehas at least 95% identity to the peptide fragment of a).

In another embodiment the peptide compound is selected from c) afunctional analogue wherein at least one amino acid has been deleted,inserted and/or substituted in SEQ ID NO 1 or the peptide fragment ofa), wherein the C-terminal amino acid also comprises the amide; or apharmaceutically acceptable salt thereof.

In a further embodiment the peptide compound is selected from a) apeptide fragment of SEQ ID NO 1 consisting of a consecutive sequence offrom 8 to 321 amino acids, wherein the C-terminal amino acid alsocomprises the amide; or a pharmaceutically acceptable salt thereof. In afurther embodiment the peptide fragment consists of a consecutivesequence in the range of from 8 to 300 amino acids. In a furtherembodiment the peptide fragment consists of a consecutive sequence inthe range of from 8 to 250 amino acids. In a further embodiment thepeptide fragment consists of a consecutive sequence in the range of from8 to 200 amino acids. In a further embodiment the peptide fragmentconsists of a consecutive sequence in the range of from 8 to 150 aminoacids. In a further embodiment the peptide fragment consists of aconsecutive sequence in the range of from 8 to 120 amino acids. In afurther embodiment the peptide fragment consists of a consecutivesequence in the range of from 10 to 100 amino acids. In a furtherembodiment the peptide fragment consists of a consecutive sequence inthe range of from 20 to 80 amino acids. In a further embodiment thepeptide fragment consists of a consecutive sequence in the range of from30 to 60 amino acids. In a further embodiment the peptide fragmentconsists of a consecutive sequence in the range of from 40 to 50 aminoacids. In a still further embodiment the peptide fragment of SEQ ID NO 1is selected from the group consisting of ARG(1-310), ARG(1-301),ARG(1-291), ARG(11-322), ARG(21-322), ARG(30-322), ARG(40-322),ARG(11-310), ARG(11-301), ARG(11-291), ARG(21-310), ARG(21-301),ARG(21-291), ARG(30-310), ARG(30-301), ARG(30-291), ARG(40-310),ARG(40-301), and ARG(40-291). In a still further embodiment the peptidefragment of SEQ ID NO 1 is selected from ARG(1-310). In a still furtherembodiment the peptide fragment of SEQ ID NO 1 is selected fromARG(1-301). In a still further embodiment the peptide fragment of SEQ IDNO 1 is selected from ARG(1-291). In a still further embodiment thepeptide fragment of SEQ ID NO 1 is selected from ARG(11-322). In a stillfurther embodiment the peptide fragment of SEQ ID NO 1 is selected fromARG(21-322). In a still further embodiment the peptide fragment of SEQID NO 1 is selected from ARG(30-322). In a still further embodiment thepeptide fragment of SEQ ID NO 1 is selected from ARG(40-322). In a stillfurther embodiment the peptide fragment of SEQ ID NO 1 is selected fromARG(11-310). In a still further embodiment the peptide fragment of SEQID NO 1 is selected from ARG(11-301). In a still further embodiment thepeptide fragment of SEQ ID NO 1 is selected from ARG(11-291). In a stillfurther embodiment the peptide fragment of SEQ ID NO 1 is selected fromARG(21-310). In a still further embodiment the peptide fragment of SEQID NO 1 is selected from ARG(21-301). In a still further embodiment thepeptide fragment of SEQ ID NO 1 is selected from ARG(21-291). In a stillfurther embodiment the peptide fragment of SEQ ID NO 1 is selected fromARG(30-310). In a still further embodiment the peptide fragment of SEQID NO 1 is selected from ARG(30-301). In a still further embodiment thepeptide fragment of SEQ ID NO 1 is selected from ARG(30-291). In a stillfurther embodiment the peptide fragment of SEQ ID NO 1 is selected fromARG(40-310). In a still further embodiment the peptide fragment of SEQID NO 1 is selected from ARG(40-301). In a still further embodiment thepeptide fragment of SEQ ID NO 1 is selected from ARG(40-291). In a stillfurther embodiment the peptide fragment of SEQ ID NO 1 is selected fromARG(161-190). In a still further embodiment the peptide fragment of SEQID NO 1 is selected from ARG(181-210). In a still further embodiment thepeptide fragment of SEQ ID NO 1 is selected from ARG(161-210).

It is to be understood that when the peptide fragment consists of aconsecutive sequence in the range of from 8 to 120, it may at the sametime be selected within the sequence of for instance ARG(40-291),whereas a peptide fragment consisting of a consecutive sequence in therange of from 8 to 321, cannot be at the same time be selected withinthe sequence of for instance ARG(40-291), this is known to the personskilled in the art. Otherwise all combinations are contemplated withinthe present invention.

It is also to be understood that ARG(x-y), wherein x and y are integersselected from 1-322 as used herein means a peptide fragment of Arginase1 having the SEQ ID NO 1 as defined herein, wherein x is the N-terminalamino acid and y is the C-terminal amino acid, for instance ARG(174-182)indicates the peptide fragment from amino acid 174 of SEQ ID NO 1 toamino acid 182 of SEQ ID NO 1 wherein amino acid 174 is I and amino acid182 is V.

The term “identity” as used herein refers to a relationship between thesequences of two or more peptides, such as polypeptides, as determinedby comparing the sequences. In the art, “identity” also means the degreeof sequence relatedness between proteins or polypeptides, as determinedby the number of matches between strings of two or more amino acidresidues. “Identity” measures the percent of identical matches betweenthe smaller of two or more sequences with gap alignments (if any)addressed by a particular mathematical model or computer program (i.e.,“algorithms”). Identity of related proteins or peptides can be readilycalculated by known methods. Such methods include, but are not limitedto, those described in Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Pro-jects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Se-quence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press,New York, 1991; and Carillo et al., SIAM J. Applied Math., 48, 1073,(1988).

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity aredescribed in publicly available computer programs. Preferred computerprogram methods to determine identity between two sequences include theGCG program package, including GAP (Devereux et al., Nucl. Acid. Res.,12, 387, (1984); Genetics Computer Group, University of Wisconsin,Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol.Biol., 215, 403-410, (1990)). The BLASTX program is publicly availablefrom the National Center for Biotechnology Information (NCBI) and othersources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894;Altschul et al., supra). The well known Smith Waterman algorithm mayalso be used to determine identity.

For example, using the computer algorithm GAP (Genetics Computer Group,University of Wisconsin, Madison, Wis.), two proteins for which thepercent sequence identity is to be determined are aligned for optimalmatching of their respective amino acids (the “matched span”, asdetermined by the algorithm). A gap opening penalty (which is calculatedas 3 times the average diagonal; the “average diagonal” is the averageof the diagonal of the comparison matrix being used; the “diagonal” isthe score or number assigned to each perfect amino acid match by theparticular comparison matrix) and a gap extension penalty (which isusually 1/10times the gap opening penalty), as well as a comparisonmatrix such as PAM 250 or BLOSUM 62 are used in conjunction with thealgorithm. A standard comparison matrix (see Dayhoff et al., Atlas ofProtein Sequence and Structure, vol. 5, supp.3 (1978) for the PAM 250comparison matrix; Henikoff et al., Proc. Natl. Acad. Sci USA, 89,10915-10919, (1992) for the BLOSUM 62 comparison matrix) is also used bythe algorithm. Preferred parameters for a protein or peptide sequencecomparison include the following: Algorithm: Needleman et al., J. Mol.Biol, 48, 443-453, (1970); Comparison matrix: BLOSUM 62 from Henikoff etal., Proc. Natl. Acad. Sci. USA, 89, 10915-10919, (1992); Gap Penalty:12, Gap Length Penalty: 4, Threshold of Similarity: 0. The GAP programis useful with the above parameters. The aforementioned parameters arethe default parameters for protein comparisons (along with no penaltyfor end gaps) using the GAP algorithm.

In a further embodiment the arginase activity is reduced compared toArginase 1 as measured by an arginase activity assay. In one embodimentthe arginase activity is reduced to inactivity. In another embodimentthe arginase activity assay is selected from the Arginase ActivityColorimetric Assay Kit (BioVision Arginase assay # K755-100). In afurther embodiment the arginase activity is reduced to inactivitycompared to Arginase 1 as measured by the arginase activity assay isselected from the Arginase Activity Colorimetric Assay Kit.

In a still further embodiment the consecutive sequence comprises one ormore sequences selected from any one of SEQ ID NO 2-17. In oneembodiment the consecutive sequence comprises the sequence selected fromSEQ ID NO 9. In another embodiment the consecutive sequence comprisesonly the sequence selected from SEQ ID NO 9, but not any one of the SEQID NO 2-8 or 10-17.

In a further embodiment the consecutive sequence comprises the sequencehaving SEQ ID NO 50. In a still further embodiment the consecutivesequence comprises the sequence having SEQ ID NO 51. In a furtherembodiment the consecutive sequence comprises the sequence having SEQ IDNO 52.

In a further embodiment the peptide fragment under a), the functionalhomologue under b), or the functional analogue under c) activates Tcells that recognizes Arginase 1 expressing cells. In one embodiment theactivation is determined by the ELISPOT assay described herein. In afurther embodiment the peptide fragment under a), activates T cells thatrecognizes Arginase 1 expressing cells as determined by the ELISPOTassay described herein.

In a particular aspect the disclosure concerns a peptide compound ofArginase 1 selected from:

a) SEQ ID NO 52 or a peptide fragment of SEQ ID NO 52 consisting of aconsecutive sequence having from 8 to 49 amino acids,

b) a functional homologue having at least 70%, 80%, 90%, or 95% identityto SEQ ID NO 52 or the peptide fragment of a), and

c) a functional analogue wherein at least one amino acid has beendeleted, inserted and/or substituted in SEQ ID NO 52 or the peptidefragment of a),

and wherein the C-terminal amino acid of any one of a), b) or c) alsocomprises the amide; or a pharmaceutically acceptable salt thereof. Itis intended that any one of the peptide compounds of a), b) or c) can bethe subject of individual embodiments.

In a further embodiment the peptide fragment consists of a consecutivesequence in the range of from 9 to 49 amino acids, such as from 10 to 40amino acids, such as from 20 to 30 amino acids.

In a further embodiment the peptide fragment of SEQ ID NO 52 consistingof a consecutive sequence having from 8 to 49 amino acids, comprises theSEQ ID NO 51. In a further embodiment the peptide fragment of SEQ ID NO52 consisting of a consecutive sequence having from 8 to 49 amino acids,comprises the SEQ ID NO 50. In a further embodiment the peptide fragmentof SEQ ID NO 52 consisting of a consecutive sequence having from 8 to 49amino acids, comprises the SEQ ID NO 9.

In a further embodiment the peptide fragment comprises at least one CD4+and at least one CD8+ T cell epitope. In a still further embodiment thepeptide fragment comprises at least one CD4+ or at least one CD8+ T cellepitope. In a further embodiment the peptide fragment comprises at leastone CD4+ T cell epitope. In a still further embodiment the peptidefragment comprises at least one CD8+ T cell epitope. In a furtherembodiment the peptide fragment comprises all T cell epitopes, inparticular all CD4+ and CD8+ epitopes, located in a hot-spot region inARG(161-210). In a still further embodiment the peptide fragmentcomprises all T cell epitopes except one, in particular all CD4+ andCD8+ epitopes, located in a hot-spot region in ARG(161-210).

In a further aspect the disclosure relates to a nucleic acid encodingthe peptide compound of the present invention. The peptide compound isselected from any one of the above embodiments. In one embodiment thenucleic acid is selected from the group consisting of DNA and RNA.

In a still further aspect the disclosure relates to a vector comprisingthe nucleic acid of the present invention. The nucleic acid is selectedfrom any one of the above embodiments, and the peptide compound of thepresent invention is selected from any one of the above embodiments. Inone embodiment the vector is selected from a virus vector.

In a further aspect the present disclosure relates to a host cellcomprising the vector of the present invention. The vector is selectedfrom any one of the above embodiments, the nucleic acid is selected fromany one of the above embodiments, and the peptide compound is selectedfrom any one of the above embodiments. In one embodiment the host cellis selected from a mammalian cell.

In a still further aspect the disclosure relates to a vector comprisinga nucleic acid encoding an inactive sequence of Arginase 1 selectedfrom:

a) a peptide fragment of SEQ ID NO 1 consisting of a consecutivesequence of from 8 to 321 amino acids,

b) a functional homologue having at least 70%, 80%, 90%, or 95% identityto SEQ ID NO 1 or the peptide fragment of a), and

c) a functional analogue wherein at least one amino acid has beendeleted, inserted and/or substituted in SEQ ID NO 1 or the peptidefragment of a),

and wherein the C-terminal amino acid of any one of a), b) or c) alsocomprises the amide, wherein the nucleic acid expresses the inactivesequence, and wherein the inactive sequence comprises at least 1immunogenic epitope. In one embodiment the epitope is selected from thegroup consisting of SEQ ID NO 2-17, typically the epitope is selectedfrom SEQ ID NO 9. In another embodiment the epitope may comprise anysequence of at least 8 consecutive amino acids comprised within any oneof SEQ ID NOs: 50-52.

In a further aspect the disclosure relates to a composition comprisingthe peptide compound or the nucleic acid or the vector or the host cellof the present disclosure, optionally together with a pharmaceuticallyacceptable additive, such as carrier or adjuvants.

In a still further aspect the present disclosure relates to animmunotherapeutic composition comprising

a) the peptide compound of the present invention or the nucleic acid ofthe present invention or the vector of the present invention or the hostcell of the present invention; and

b) an adjuvant;

for use as a medicament.

In an embodiment the immunotherapeutic composition is for use in amethod for treatment or prevention of a disease, disorder or conditionselected from cancer. In one embodiment the cancer is a tumor formingcancer disease. In a further embodiment the adjuvant is selected fromthe group consisting of bacterial DNA based adjuvants, oil/surfactantbased adjuvants, viral dsRNA based adjuvants, imidazochinilines, and aMontanide ISA adjuvant.

In a further aspect the present disclosure relates to a kit-of-partscomprising;

a) the immunotherapeutic composition of the present invention, and

b) a composition comprising at least one second active ingredient,selected from an immunostimulating compound, such as an interleukin,e.g. IL-2 and or IL-21, an anti-cancer agent, such as Actimide,Azacitidine, Azathio-prine, Bleomycin, Carboplatin, Capecitabine,Cisplatin, Chlorambucil, Cyclophospha-mide, Cytarabine, Daunorubicin,Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Etoposide,Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin,Irinotec-an, Lenalidomide, Leucovorin, Mechlorethamine, Melphalan,Mercaptopurine, Metho-trexate, Mitoxantrone, nivolumab, Oxaliplatin,Paclitaxel, pembrolizumab, Pemetrexed, Revlimid, Temozolomide,Teniposide, Thioguanine, Valrubicin, Vinblastine, Vincristine, Vindesineand Vinorelbine.

In an embodiment of the kits-of-parts, the provided compositions are tobe administered simultaneously or sequentially.

In a further aspect the disclosure relates to a method of treating aclinical condition characterized by expression of Arginase 1 of SEQ IDNO 1, the method comprising administering to an individual sufferingfrom said clinical condition an effective amount of the peptide compoundof the present invention or the nucleic acid of the present invention orthe vector of the present invention or the host cell of the presentinvention.

In a further aspect the disclosure relates to a method of stimulation ofarginase 1 specific T-cells, such as CD4 and CD8 T-cells, in a cancerpatient, the method comprising administering to the cancer patient aneffective amount of the peptide compound of, or the nucleic, or thevector, or the host cell of the disclosure.

In a still further aspect the present disclosure relates to a method ofsuppressing an immune suppressive function of Arginase 1 expressingcells, in a cancer patient, the method comprising administering to thecancer patient an effective amount of the peptide compound, or thenucleic acid, or the vector, or the host cell of the disclosure.

In a further aspect the disclosure relates to use of the peptidecompound, or the nucleic acid, or the vector, or the host cell of thedisclosure, for the manufacture of a medicament, such as animmunotherapeutic composition or vaccine, for the treatment orprevention of a cancer characterized by expression of Arginase 1.

In a still further aspect the disclosure relates to the peptidecompound, or the nucleic acid, or the vector, or the host cell of thedisclosure, for use in a method for treatment or prevention of a cancer,when administered simultaneously or sequentially with an additionalcancer therapy.

The additional cancer therapy is selected from the group consisting of acytokine therapy, a T-cell therapy, an NK therapy, an immune systemcheckpoint inhibitor, chemotherapy, radiotherapy, immunostimulatingsubstances, gene therapy, anti-bodies and dendritic cells. In oneembodiment the additional cancer therapy is selected from an immunesystem checkpoint inhibitor, such as a checkpoint blocking antibody (e.gnivolumab, pembrolizumab), or is selected from the group consisting ofActimide, Azacitidine, Azathioprine, Bleomycin, Carboplatin,Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine,Dauno-rubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin,Etoposide, Fludarabine, Fluor-ouracil, Gemcitabine, Hydroxyurea,Idarubicin, Irinotecan, Lenalidomide, Leucovorin, Mechlorethamine,Melphalan, Mercaptopurine, Methotrexate, Mitoxantrone, Oxaliplatin,Paclitaxel, Pemetrexed, Revlimid, Temozolomide, Teniposide, Thioguanine,Valrubicin, Vinblastine, Vincristine, Vindesine and Vinorelbine.

Arginase activity may be measured for instance by using the ArginaseActivity Colorimetric Assay Kit (BioVision Arginase assay # K755-100).BioVision's Arginase Activity Assay kit is simple, sensitive and rapid.In this assay, Arginase reacts with arginine & undergoes a series ofreactions to form an intermediate that reacts stoichiometrically withOxiRed™ Probe to generate the colored product (OD 570 nm). The kit candetect Arginase activity less than 0.2 U/L in 96-well assay format.

As used herein any amino acid sequence shown may be modified at theC-terminal amino acid to be in amide form (—CONH₂) or may be in acidform (—COOH), thus any one of these are preferred embodiments, and it isintended that any C-terminal amino acid, such as I, F, R, L, K, G, M, D,V, S, T, N, Y, P comprises both amide and acid forms unless specified by—NH₂ or —OH.

The arginase peptide fragments disclosed herein are made by standardpeptide synthesis, such as solid-phase peptide synthesis (SPPS). SPPS isa standard method for synthesizing peptides in the lab. SPPS allows forthe synthesis of natural peptides which are difficult to express inbacteria, the incorporation of unnatural amino acids, peptide/proteinbackbone modification, and the synthesis of D-proteins, which consist ofD-amino acids. Small porous beads are treated with functional units(‘linkers’) on which peptide chains can be built. The peptide willremain covalently attached to the bead until cleaved from it by areagent such as anhydrous hydrogen fluoride or trifluoroacetic acid. Thepeptide is thus ‘immobilized’ on the solid-phase and can be retainedduring a filtration process while liquid-phase reagents and by-productsof synthesis are flushed away. The general principle of SPPS is one ofrepeated cycles of deprotection-wash-coupling-wash. The free N-terminalamine of a solid-phase attached peptide is coupled to a singleN-protected amino acid unit. This unit is then deprotected, revealing anew N-terminal amine to which a further amino acid may be attached. Thesuperiority of this technique partially lies in the ability to performwash cycles after each reaction, removing excess reagent with all of thegrowing peptide of interest remaining covalently attached to theinsoluble resin. There are two majorly used forms of SPPS-Fmoc and Boc.Unlike ribosome protein synthesis, solid-phase peptide synthesisproceeds in a C-terminal to N-terminal fashion. The N-termini of aminoacid monomers is protected by either of these two groups and added ontoa deprotected amino acid chain. Automated synthesizers are available forboth techniques, though many research groups continue to perform SPPSmanually. Furthermore, the skilled person will understand that theprocesses described above and hereinafter the functional groups ofintermediate compounds may need to be protected by protecting group.

When the peptide compounds, nucleic acids, vectors, host cells andpharmaceutical compositions herein disclosed are used for the abovetreatment, a therapeutically effective amount of at least one compoundis administered to a mammal in need of said treatment.

As used herein amino acids are identified by the one or three lettercode known to the person skilled in the art and shown in the table belowfor convenience:

Amino acids, one and three letter codes Three One Amino acid letter codeletter code alanine ala A arginine arg R asparagine asn N aspartic acidasp D asparagine or asx B aspartic acid cysteine cys C glutamic acid gluE glutamine gln Q glutamine or glx Z glutamic acid glycine gly Ghistidine his H isoleucine ile I leucine leu L lysine lys K methioninemet M phenylalanine phe F proline pro P serine ser S threonine thr Ttryptophan trp W tyrosine tyr Y valine val V

The term “treatment” and “treating” as used herein means the managementand care of a patient for the purpose of combating a condition, such asa disease or a disorder. The term is intended to include the fullspectrum of treatments for a given condition from which the patient issuffering, such as administration of the active compound to alleviatethe symptoms or complications, to delay the progression of the disease,disorder or condition, to alleviate or relief the symptoms andcomplications, and/or to cure or eliminate the disease, disorder orcondition as well as to prevent the condition, wherein prevention is tobe understood as the management and care of a patient for the purpose ofcombating the disease, condition, or disorder and includes theadministration of the active compounds to prevent the onset of thesymptoms or complications. The treatment may either be performed in anacute or in a chronic way. The patient to be treated is preferably amammal; in particular a human being, but it may also include animals,such as dogs, cats, cows, monkeys, apes, sheep and pigs.

The term “a therapeutically effective amount” of a peptide compound ofthe present invention or a peptide fragment disclosed herein, as usedherein means an amount sufficient to cure, alleviate or partially arrestthe clinical manifestations of a given disease and its complications. Anamount adequate to accomplish this is defined as “therapeuticallyeffective amount”. Effective amounts for each purpose will depend on theseverity of the disease or injury as well as the weight and generalstate of the subject. It will be understood that determining anappropriate dosage may be achieved using routine experimentation, byconstructing a matrix of values and testing different points in thematrix, which is all within the ordinary skills of a trained physicianor veterinary.

In a still further aspect the disclosure relates to a pharmaceuticalcomposition comprising the peptide compound, such as peptide fragment,of the present invention and optionally a pharmaceutically acceptableadditive, such as a carrier or an excipient.

As used herein “pharmaceutically acceptable additive” is intendedwithout limitation to include carriers, excipients, diluents, adjuvant,colorings, aroma, preservatives etc. that the skilled person wouldconsider using when formulating a compound of the present invention inorder to make a pharmaceutical composition.

The adjuvants, diluents, excipients and/or carriers that may be used inthe composition of the invention must be pharmaceutically acceptable inthe sense of being compatible with the peptide compound, peptidefragment, nucleic acid, vector, or host cell and the other ingredientsof the pharmaceutical composition, and not deleterious to the recipientthereof. It is preferred that the compositions shall not contain anymaterial that may cause an adverse reaction, such as an allergicreaction. The adjuvants, diluents, excipients and carriers that may beused in the pharmaceutical composition of the invention are well knownto a person within the art.

Adjuvants are any substance whose admixture into the compositionincreases or otherwise modifies the immune response elicited by thecomposition. Adjuvants, broadly defined, are substances which promoteimmune responses. Adjuvants may also preferably have a depot effect, inthat they also result in a slow and sustained release of an active agentfrom the administration site. A general discussion of adjuvants isprovided in Goding, Monoclonal Antibodies: Principles & Practice (2ndedition, 1986) at pages 61-63.

Adjuvants may be selected from the group consisting of: AlK(SO4)2,AlNa(SO4)2, AlNH4 (SO4), silica, alum, Al(OH)3, Ca3 (PO4)2, kaolin,carbon, aluminum hydroxide, muramyl dipeptides,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP),N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred toas nor-MDP),N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′2′-dipalmitoyl-sn-glycero-3-hydroxphosphoryloxy)-ethylamine(CGP 19835A, also referred to as MTP-PE), RIBI (MPL+ TDM+CWS) in a 2%squalene/Tween-80® emulsion, lipopolysaccharides and its variousderivatives, including lipid A, Freund's Complete Adjuvant (FCA),Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (forexample, poly IC and poly AU acids), wax D from Mycobacterium,tuberculosis, substances found in Corynebacterium parvum, Bordetellapertussis, and members of the genus Brucella, Titermax, ISCOMS, Quil A,ALUN (see U.S. Pat. No. 58,767 and U.S. Pat. No. 5,554,372), Lipid Aderivatives, choleratoxin derivatives, HSP derivatives, LPS derivatives,synthetic peptide matrixes or GMDP, Interleukin 1, Interleukin 2,Montanide ISA-51 and QS-21. Various saponin extracts have also beensuggested to be useful as adjuvants in immunogenic compositions.Granulocyte-macrophage colony stimulating factor (GM-CSF) may also beused as an adjuvant.

Preferred adjuvants to be used with the invention include oil/surfactantbased adjuvants such as Montanide adjuvants (available from Seppic,Belgium), preferably Montanide ISA-51. Other preferred adjuvants arebacterial DNA based adjuvants, such as adjuvants including CpGoligonucleotide sequences. Yet other preferred adjuvants are viral dsRNAbased adjuvants, such as poly I:C. GM-CSF and Imidazochinilines are alsoexamples of preferred adjuvants.

The adjuvant is most preferably a Montanide ISA adjuvant. The MontanideISA adjuvant is preferably Montanide ISA 51 or Montanide ISA 720.

In Goding, Monoclonal Antibodies: Principles & Practice (2nd edition,1986) at pages 61-63 it is also noted that, when an antigen of interestis of low molecular weight, or is poorly immunogenic, coupling to animmunogenic carrier is recommended. A peptide compound, peptidefragment, nucleic acid, vector, or host cell of an immunotherapeuticcomposition of the invention may be coupled to a carrier. A carrier maybe present independently of an adjuvant. The function of a carrier canbe, for example, to increase the molecular weight of the peptidecompound, peptide fragment, nucleic acid, vector, or host cell in orderto increase activity or immunogenicity, to confer stability, to increasethe biological activity, or to increase serum half-life. Furthermore, acarrier may aid in presenting the polypeptide or fragment thereof toT-cells. Thus, in the immunogenic composition, the polypeptide orfragment thereof may be associated with a carrier such as those set outbelow.

The carrier may be any suitable carrier known to a person skilled in theart, for example a protein or an antigen presenting cell, such as adendritic cell (DC). Carrier proteins include keyhole limpet hemocyanin,serum proteins such as transferrin, bovine serum albumin, human serumalbumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, suchas insulin or palmitic acid. Alternatively, the carrier protein may betetanus toxoid or diphtheria toxoid. Alternatively, the carrier may be adextran such as sepharose. The carrier must be physiologicallyacceptable to humans and safe.

The immunotherapeutic composition may optionally comprise apharmaceutically acceptable excipient. The excipient must be‘acceptable’ in the sense of being compatible with the other ingredientsof the composition and not deleterious to the recipient thereof.Auxiliary substances, such as wetting or emulsifying agents, pHbuffering substances and the like, may be present in the excipient.These excipients and auxiliary substances are generally pharmaceuticalagents that do not induce an immune response in the individual receivingthe composition, and which may be administered without undue toxicity.Pharmaceutically acceptable excipients include, but are not limited to,liquids such as water, saline, polyethyleneglycol, hyaluronic acid,glycerol and ethanol. Pharmaceutically acceptable salts can also beincluded therein, for example, mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like; andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. A thorough discussion of pharmaceuticallyacceptable excipients, vehicles and auxiliary substances is available inRemington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

The immunotherapeutic composition may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable compositions may be prepared, packaged, orsold in unit dosage form, such as in ampoules or in multi-dosecontainers containing a preservative. Compositions include, but are notlimited to, suspensions, solutions, emulsions in oily or aqueousvehicles, pastes, and implantable sustained-release or biodegradableformulations. In one embodiment of a composition, the active ingredientis provided in dry (for e.g., a powder or granules) form forreconstitution with a suitable vehicle (e. g., sterile pyrogen-freewater) prior to administration of the reconstituted composition. Thecomposition may be prepared, packaged, or sold in the form of a sterileinjectable aqueous or oily suspension or solution. This suspension orsolution may be formulated according to the known art, and may comprise,in addition to the active ingredient, additional ingredients such as theadjuvants, excipients and auxiliary substances described herein. Suchsterile injectable formulations may be prepared using a non-toxicparenterally-acceptable diluent or solvent, such as water or 1,3-butanediol, for example. Other acceptable diluents and solvents include, butare not limited to, Ringer's solution, isotonic sodium chloridesolution, and fixed oils such as synthetic mono- or di-glycerides.

Other compositions which are useful include those which comprise theactive ingredient in microcrystalline form, in a liposomal preparation,or as a component of a biodegradable polymer systems. Compositions forsustained release or implantation may comprise pharmaceuticallyacceptable polymeric or hydrophobic materials such as an emulsion, anion exchange resin, a sparingly soluble polymer, or a sparingly solublesalt. Alternatively, the active ingredients of the composition may beencapsulated, adsorbed to, or associated with, particulate carriers.Suitable particulate carriers include those derived from polymethylmethacrylate polymers, as well as PLG microparticles derived frompoly(lactides) and poly(lactide-co-glycolides). See, e.g., Jeffery etal. (1993) Pharm. Res. 10:362-368. Other particulate systems andpolymers can also be used, for example, polymers such as polylysine,polyarginine, polyornithine, spermine, spermidine, as well as conjugatesof these molecules.

As mentioned above, the compositions and particularly immunotherapeticcompositions as herein disclosed may, in addition to the compoundsherein disclosed, further comprise at least one pharmaceuticallyacceptable adjuvant, diluent, excipient and/or carrier. In someembodiments, the pharmaceutical compositions comprise from 1 to 99weight % of said at least one pharmaceutically acceptable adjuvant,diluent, excipient and/or carrier and from 1 to 99 weight % of acompound as herein disclosed. The combined amount of the activeingredient and of the pharmaceutically acceptable adjuvant, diluent,excipient and/or carrier may not constitute more than 100% by weight ofthe composition, particularly the pharmaceutical composition.

In some embodiments, only one compound as herein disclosed is used forthe purposes discussed above.

In some embodiments, two or more of the compound as herein disclosed areused in combination for the purposes discussed above.

The composition, particularly immunotherapetic composition comprising acompound set forth herein may be adapted for oral, intravenous, topical,intraperitoneal, nasal, buccal, sublingual, or subcutaneousadministration, or for administration via the respiratory tract in theform of, for example, an aerosol or an air-suspended fine powder.Therefore, the pharmaceutical composition may be in the form of, forexample, tablets, capsules, powders, nanoparticles, crystals, amorphoussubstances, solutions, transdermal patches or suppositories.

Further embodiments of the process are described in the experimentalsection herein, and each individual process as well as each startingmaterial constitutes embodiments that may form part of embodiments.

The above embodiments should be seen as referring to any one of theaspects (such as ‘method for treatment’, ‘immunotherapetic composition’,‘peptide compound for use as a medicament’, or ‘peptide compound for usein a method’) described herein as well as any one of the embodimentsdescribed herein unless it is specified that an embodiment relates to acertain aspect or aspects of the present invention.

All references, including publications, patent applications and patents,cited herein are hereby incorporated by reference to the same extent asif each reference was individually and specifically indicated to beincorporated by reference and was set forth in its entirety herein.

All headings and sub-headings are used herein for convenience only andshould not be construed as limiting the invention in any way.

Any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

It is to be understood that different applications of the disclosedproducts and methods may be tailored to the specific needs in the art.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the invention only, andis not intended to be limiting.

In addition as used in this specification and the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “apeptide” includes two or more such peptides.

A “polypeptide” is used herein in its broadest sense to refer to acompound of two or more subunit amino acids, amino acid analogs, orother peptidomimetics. The term “polypeptide” thus includes shortpeptide sequences and also longer polypeptides and proteins. As usedherein, the term “amino acid” refers to either natural and/or unnaturalor synthetic amino acids, including both D or L optical isomers, aminoacid analogs and peptidomimetics, and any pharmaceutically acceptablesalts thereof.

A “subject” as used herein includes any mammal, preferably a human.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless other-wise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. Unless otherwise stated, all exact valuesprovided herein are representative of corresponding approximate values(e.g., all exact exemplary values provided with respect to a particularfactor or measurement can be considered to also pro-vide a correspondingapproximate measurement, modified by “about,” where appropriate).

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theinvention unless as much is explicitly stated.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability and/or enforceability of such patent documents.

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having”, “including” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

This invention includes all modifications and equivalents of the subjectmatter recited in herein to the maximum extent permitted by applicablelaw.

ASPECTS

The following are some additional aspects of the present disclosure:

-   1. A peptide compound of Arginase 1 selected from:    -   a) a peptide fragment of SEQ ID NO 1 consisting of a consecutive        sequence of from 8 to 321 amino acids,    -   b) a functional homologue having at least 70%, 80%, 90%, or 95%        identity to SEQ ID NO 1 or the peptide fragment of a), and    -   c) a functional analogue wherein at least one amino acid has        been deleted, inserted and/or substituted in SEQ ID NO 1 or the        peptide fragment of a),    -   and wherein the C-terminal amino acid of any one of a), b) or c)        also comprises the amide; or a pharmaceutically acceptable salt        thereof.-   2. The peptide compound of aspect1 selected from a) a peptide    fragment of SEQ ID NO 1 consisting of a consecutive sequence of from    8 to 321 amino acids, wherein the C-terminal amino acid also    comprises the amide;    -   or a pharmaceutically acceptable salt thereof.-   3. The peptide compound of aspect2 wherein the peptide fragment    consists of a consecutive sequence in the range of from 8 to 300    amino acids, 8 to 250 amino acids, 8 to 200 amino acids, 8 to 150    amino acids, 8 to 120 amino acids, e.g. 10 to 100 amino acids, 20 to    80 amino acids, 30 to 60 amino acids, 40 to 50 amino acids.-   4. The peptide compound of any one of aspects 2-3 wherein the    peptide fragment of SEQ ID NO 1 is selected from the group    consisting of ARG(1-310), ARG(1-301), ARG(1-291), ARG(11-322),    ARG(21-322), ARG(30-322), ARG(40-322), ARG(11-310), ARG(11-301),    ARG(11-291), ARG(21-310), ARG(21-301), ARG(21-291), ARG(30-310),    ARG(30-301), ARG(30-291), ARG(40-310), ARG(40-301), and ARG(40-291).-   5. The peptide compound of any one of aspects 1-4 wherein the    arginase activity is reduced compared to Arginase 1, preferably    reduced to inactivity, as measured by an arginase activity assay,    such as the Arginase Activity Colorimetric Assay Kit (BioVision    Arginase assay # K755-100).-   6. The peptide compound of any one of aspects 2-5 wherein the    consecutive sequence comprises one or more sequences selected from    any one of SEQ ID NO 2-17, such as one sequence selected from SEQ ID    NO 9.-   7. The peptide compound of any one of aspects 1-6 wherein the    peptide fragment under a), the functional homologue under b), or the    functional analogue under c) activates T cells that recognizes    Arginase 1 expressing cells.-   8. The peptide compound of aspect7 wherein the activation is    determined by the ELISPOT assay described herein.-   9. A nucleic acid, such as DNA or RNA, encoding the peptide compound    of any one of the preceding aspects.-   10. A vector, such virus vector, comprising the nucleic acid of    aspect9.-   11. A host cell, such as mammalian cell, comprising the vector of    aspect10.-   12. A vector comprising a nucleic acid encoding an inactive sequence    of Arginase 1 selected from:    -   a) a peptide fragment of SEQ ID NO 1 consisting of a consecutive        sequence of from 8 to 321 amino acids,    -   b) a functional homologue having at least 70%, 80%, 90%, or 95%        identity to SEQ ID NO 1 or the peptide fragment of a), and    -   c) a functional analogue wherein at least one amino acid has        been deleted, inserted and/or substituted in SEQ ID NO 1 or the        peptide fragment of a),    -   and wherein the C-terminal amino acid of any one of a), b) or c)        also comprises the amide, wherein the nucleic acid expresses the        inactive sequence, and wherein the inactive sequence comprises        at least 1 immunogenic epitope, such as an epitope selected from        the group consisting of SEQ ID NO 2-17, typically the epitope is        selected from SEQ ID NO 9.-   13. A composition comprising the peptide compound of any one of    aspects 1-8 or the nucleic acid of aspect 9 or vector of aspect10 or    12 or host cell of aspect11, optionally together with a    pharmaceutically acceptable additive, such as carrier or adjuvants.-   14. An immunotherapeutic composition comprising    -   a) the peptide compound of any one of aspects 1-8 or the nucleic        acid of aspect9 or vector of aspect10 or 12 or host cell of        aspect11; and    -   b) an adjuvant;    -   for use as a medicament.-   15. The immunotherapeutic composition of aspect14 for use in a    method for treatment or prevention of a disease, disorder or    condition selected from cancer, such as a tumor forming cancer    disease.-   16. The immunotherapeutic composition of any one of aspects 14-15    wherein the adjuvant is selected from the group consisting of    bacterial DNA based adjuvants, oil/surfactant based adjuvants, viral    dsRNA based adjuvants, imidazochinilines, a Montanide ISA adjuvant.-   17. A kit-of-parts comprising;    -   a) the immunotherapeutic composition of any one of aspects        14-16, and    -   b) a composition comprising at least one second active        ingredient, selected from an immunostimulating compound, such as        an interleukin, e.g. IL-2 and or IL-21, an anti-cancer agent,        such as a chemotherapeutic agent, e.g. Actimide, Azacitidine,        Azathioprine, Bleomycin, Carboplatin, Capecitabine, Cisplatin,        Chlorambucil, Cyclophosphamide, Cytarabine, Daunorubicin,        Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Etoposide,        Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin,        Irinotecan, Lenalidomide, Leucovorin, Mechlorethamine,        Melphalan, Mercaptopurine, Methotrexate, Mitoxantrone,        nivolumab, Oxaliplatin, Paclitaxel, pembrolizumab, Pemetrexed,        Revlimid, Temozolomide, Teniposide, Thioguanine, Valrubicin,        Vinblastine, Vincristine, Vindesine and Vinorelbine.-   18. The kits-of-parts according to aspect17, where the provided    compositions are to be administered simultaneously or sequentially.-   19. A method of treating a clinical condition characterized by    expression of Arginase 1 of SEQ ID NO 1, the method comprising    administering to an individual suffering from said clinical    condition an effective amount of the peptide compound of any one of    aspects 1-8 or the nucleic acid of aspect9 or vector of aspect10 or    12 or host cell of aspect 11.-   20. A method of stimulation of arginase 1 specific T-cells, such as    CD4 and CD8 T-cells, in a cancer patient, the method comprising    administering to the cancer patient an effective amount of the    peptide compound of any one of aspects 1-8 or the nucleic acid of    aspect9 or vector of aspect10 or 12 or host cell of aspect 11.-   21. A method of suppressing an immune suppressive function of    Arginase 1 expressing cells, in a cancer patient, the method    comprising administering to the cancer patient an effective amount    of the peptide compound of any one of aspects 1-8 or the nucleic    acid of aspect9 or vector of aspect10 or 12 or host cell of aspect    11.-   22. Use of the peptide compound of any one of aspects 1-8 or the    nucleic acid of aspect9 or vector of aspect10 or 12 or host cell of    aspect 11 for the manufacture of a medicament, such as an    immunotherapeutic composition or vaccine, for the treatment or    prevention of a cancer characterized by expression of Arginase 1.-   23. A peptide compound of any one of aspects 1-8 or the nucleic acid    of aspect9 or vector of aspect10 or 12 or host cell of aspect 11,    for use in a method for treatment or prevention of a cancer, when    administered simultaneously or sequentially with an additional    cancer therapy, such as a cytokine therapy, a T-cell therapy, an NK    therapy, an immune system checkpoint inhibitor, chemotherapy,    radiotherapy, immunostimulating substances, gene therapy, antibodies    and dendritic cells.-   24. The peptide fragment, nucleic acid, vector or host cell of    aspect23 wherein the checkpoint blocking antibodies are selected    from Actimide, Azacitidine, Azathioprine, Bleomycin, Carboplatin,    Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine,    Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin,    Etoposide, Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea,    Idarubicin, Irinotecan, Lenalidomide, Leucovorin, Mechlorethamine,    Melphalan, Mercaptopurine, Methotrexate, Mitoxantrone, Nivolumab,    Oxaliplatin, Paclitaxel, Pembrolizumab, Pemetrexed, Revlimid,    Temozolomide, Teniposide, Thioguanine, Valrubicin, Vinblastine,    Vincristine, Vindesine and Vinorelbine.

The present invention is further illustrated by the following examplesthat, however, are not to be construed as limiting the scope ofprotection. The features disclosed in the foregoing description and inthe following examples may, both separately and in any combinationthereof, be material for realizing the invention in diverse formsthereof.

EXAMPLES

Introduction

The following explains the rationale behind the planning and executionof some of the following experiments:

In addition to the new immunogenic epitopes from Arginase 1, and strongimmune responses against the new immunogenic epitopes as well asfrequent immune responses detected against several peptide fragments ofArginase 1, the present inventor also will identify if T cells likewiserecognize Arginase. Furthermore, immunogenic epitopes from Arginase willbe identified. Using the reverse immunology approach, potential MHCclass I binding motifs will be identified followed by the synthesis ofcandidate peptides. Subsequently, the actual binding to thecorresponding MHC alleles will be established. Following theidentification of class I binding peptide compounds, such peptides willbe examined in an ELISPOT assay for cytokine release by peptide specificT cells in order to examine whether specific T cells are present amongperipheral PBMC of cancer patients as well as healthy donors. In theplanned study tetrameric MHC/-peptide-complexes will be used asadditional means of investigating T-cell reactivity in peripheral bloodby flow cytometry. Finally, tetrameric MHC/peptide complexes will beused to isolate specific T cells directly from patient blood or tumorinfiltrated lymph nodes. This will be used determine the functionalcapacity of specific T cells.

Additionally, the inventor will search for novel epitopes presented byMHC class II molecules, since CD4+ T cells play a critical role ingenerating and maintaining antigen-specific cellular and humoral immuneresponses. CD4+ T cells are presented with 18-mer overlapping syntheticpeptides spanning the entire protein sequence in ELISPOT assays.Peptide-specific CD4+ T-cell clones are generated by repetitivestimulation with peptide. Furthermore, using a set of partiallyhistocompatible EBV-B cell lines and MHC class II-specific antibodies,the HLA class II restriction elements will be identified. Finally, tumorinfiltrating lymphocytes (TIL) cultures will be analyzed for reactivityagainst CD4 and CD8 peptide epitopes revealing important informationregarding pivotal in vivo targets.

Arginase is a major player in the immune system and is, it could bespeculated that such T cells are involved in general immune regulation.An additional part of these studies aim at analysing the role ofArginase specific T cells may therefore also have a role in immuneregulation. First, it will be examined if Arginase-specific T cells caneffect immunity by eliminating arginase-expressing regulatory cellsthereby suppressing and/or delaying local immune suppression. To examinea possible immune effect of Argiasese T cells, it will be examined ifThe presence of Arginase-specific T cells or the activation of such Tcells by Arginase-derived peptides may boost additional T- and/or B-cellresponses against other antigens. Regulatory cells contribute to thestrength and duration of a given immune response. Thus, any “supportive”effect of Arginase-specific T cells on other immune cells may well bemediated in several direct and indirect manners. In this respect, thelevel of essential amino acids will be examined, the frequency of Tregsas well as the frequency of IL-17 producing cells. Furthermore, theeffect of Arginase-specific T cells on the overall production ofdifferent cytokines including INF-γ, IL-6, TNF-α as well as IL-10 andTGF-β, will be examined. Another possible effect of Arginase-specific Tcells could be mediated through the metabolites of arginin. Furthermore,the phenotype of the Arginase-specific T cells by FACS as well asanalysing the cytokine profile of such T cells will be described.Furthermore, the expression of co-stimulatory molecules (FACS), director indirect killing of effector cells and APC (cytotox assays), will beaddressed. The use of leukapheresis samples from cancer patientscontaining huge numbers of T cells will make it possible to performexperiments with a natural subset of Arginase-specific T cells directlyisolated from donors.

Peptides

The sequences of the peptides used in these experiments are shown infull in Table A below (see the section entitled “Sequences”). Peptidesare described in Table A by SEQ ID NO, by name, or by reference to thestart and end positions of each peptide sequence within the full lengthsequence of Arginase 1. Each may be used interchangeably. For example,the peptide of SEQ ID NO: 34 may alternatively be referred to by thename Arg1-17 or may alternatively be referred to as Arg 161-190 (given astart position of 161 and end position of 190). The intended referencein each case will be clear from the context. The peptide of SEQ ID NO: 9may additionally be referred to as ArgShort.

20-mer and 22-mer peptides were synthesized by PepScan (Netherlands) anddissolved in DMSO at 10 mM. 30-mer and 50-mer peptides were synthesizedby Schafer-N ApS (Denmark) and dissolved in DMSO at 10 mM stock. Shorterpeptides were synthesized by KJ Ross-Petersen ApS (Denmark) anddissolved in sterile water to a stock concentration of 2 mM. Purity ofthe synthesized peptides was >80%.

Example 1

Patients, Protocols and Methods

Patient Material:

The present project is based on analyses of blood and tumor lesions frommelanoma, renal cell carcinoma and ovarian cancer patients. Collectionof blood and tumor samples was conducted at Herlev Hospital. Bloodsamples were drawn a minimum of four weeks after termination of any kindof anti-cancer therapy. PBMC were isolated using Lymphoprep™ (Alere A S,cat. 1114547) separation, HLA-typed and frozen in FCS with 10% DMSO(Sigma-Aldrich, cat. D5879-100 ML). The immediate processing of thematerial was handled at the CCIT (Centre for Cancer Immunotherapy) inaccordance with local ethical requirements. All patients onlyparticipate upon written informed consent, and that the ethicalcommittee have approved the projects.

Enzyme-Linked Immunospot (ELISPOT):

Using the “ELISPOT” technique it is possible to screen for T-cellrecognition of a high number of peptide antigens despite theavailability of relatively few T-cells. The “ELISPOT” technique takesadvantage of the fact that T-cells synthesize cytokines e.g. IFN-γ uponTCR engagement and subsequent signaling. The method may be used toanalyze for secretion of any cytokine of choice—in our laboratory weroutinely use IFN-γ, TNF-α, IL-10, Granzyme-B as well asperforin—ELISPOT analyses. Standardized quantitation is accomplished bythe use of an ELISPOT reader (IMMUNOSPOT, CTLanalyzers LLChttp://www.immunospotcom/).

Cytotoxicity Assay

Conventional ⁵¹Cr-release assays for CTL-mediated cytotoxicity werecarried out as described elsewhere [3]. Target cells were T2-cells(ATCC), HLA-A²⁺ melanoma cell lines (FM3 and FM93)

FACS:

Over the past years, new strong FACS techniques have been developed—inparticular for studies of immune function and specificity. In thisrespect, peptide/HLA complexes are readily used for analyzing thefrequency of peptide specific T-cells. Specific T-cells can be isolatedand expanded in vitro. For Intracellular stainings, cells werestimulated with Arginase derived peptide and for surface markers weemployed 4 μl NIR, 10 μl CD4 PerCP, 2 μl CD8 Pacific Blue, and 10 μl CD3FITC and for intracellular staining we used 2 μl anti-TNF-a andanti-IFN-g antibodies conjugated with either PE-Cy7 or APC. Washing,permeabilization and staining procedures followed previously describedmethods [4]. Flow cytometry analysis was performed on a FACSCANTO II (BDBiosciences, San Jose Calif., USA)

Results:

16 peptides derived from Arginase 1 were designed. These peptides arereferred to herein as Arg 1 to Arg 16 (SEQ ID NOs: 12 to 17). Thesequences of these peptides are shown in full in Table A below (see thesection entitled “Sequences”). 15 of the peptides (Arg1 to Arg15) wereexamined in ELISPOT. We scrutinized peripheral blood mononuclear cells(PBMC) from six melanoma patients for the presence of specific T-cellresponses against Arg-derived peptide using the IFN-gamma ELISPOTsecretion assay. Strong responses against Arg2 (SEQ ID NO 3), Arg3 (SEQID NO 4), Arg5 (SEQ ID NO 6), Arg 6 (SEQ ID NO 7), Arg7 (SEQ ID NO 8),Arg8 (SEQ ID NO 9), Arg9 (SEQ ID NO 10), Arg 10 (SEQ ID NO 11), Arg11(SEQ ID NO 12), Arg14 (SEQ ID NO 15), Arg15 (SEQ ID NO 16) weredetected. FIG. 1 exemplifies Arg-specific T cell responses. EspeciallyArg8 (SEQ ID NO 9) was examined in more detail. Patient PBMC hostingimmune responses towards Arg8. Immune responses (specific cells/3×10e5cells) from 6 cancer patients against Arginase-derived Arg 8 peptide asseen in FIG. 2. Very strong responses were frequently detected amongpatients PBMC.

We next examined overlapping 20mer overlapping peptides spanning theentire Arginase sequence SEQ ID NO 1. Thus there are 30×20mer peptidesand 1×22mer to span the entire length of the sequence. The sequences ofthese peptides are shown in full in Table A below (see the sectionentitled “Sequences”) and correspond to SEQ ID NOs: 18 to 48. We usedthe above described peptides to examine immune responses in PMBC fromthree cancer patients seen in FIG. 3. Frequent immune responses weredetected against several peptides especially the long peptides referredto as Arg1-23, Arg1-18, Arg1-27, Arg1-17, Arg1-19, Arg1-12, Arg1-20,Arg1-4, Arg1-1, Arg1-7, Arg1-14, Arg1-15, Arg1-6, Arg1-16, Arg1-22,Arg1-29. In addition, we show responses in PBMC from healthy individualsagainst these long peptides (FIG. 4).

CD8 positive culture can kill target cells loaded with Arg8 on thesurface as examined by standard chromium release assay as seen in FIG.5. Killing of Arginase positive cancer (melanoma) cell lines FM3 andFM93 by Arginase specific T cells was also demonstrated using standardchromium release assays as seen in FIG. 6. In this experiment Arginasespecific T cells were incubated 4 h with either Cr-labeled melanoma celllines FM3 or FM93 at different effector:target ratios. Both cell linesexpress intracellular Arginase.

Next we examined if CD4 CD4 T-cells can recognize arginase after invitro stimulation. Hence, we chose to analyze PBMC from a patient with aresponse against Arg8 using intracellular cytokine staining. Althoughthis method is less sensitive than ELISPOT, it allows to elucidate whichimmune cells secrete the cytokine identified in ELISPOT. Hence, westimulated CD4 T cells from a cancer patients 5 times with Arg8 peptide.Next, we performed Intracellular staining against INF (PE-Cy7A) andTNF-alfa (APC-A) of the T-cell culture after 5 stimulations in vitrowith Arg8 peptide. The culture is either stimulated with or without Arg8peptide as seen in FIG. 7. Similar results are seen in FIG. 8.

In conclusion: Both CD8 and CD4 T cells can recognize Arginase derivedpeptides on the surface of target cells. The region spanning positions161 to 190 of Arginasel appears to be particularly immunogenic. Thisregion may be referred to herein as a hotspot.

Example 2

Materials and Methods

Additional Peptide Stimulation and ELISPOT Assay

PBMCs from healthy donors or cancer patients were stimulated with 80 μgof arginase-derived peptides and 120 U/ml IL-2 (Peprotech, London, UK,cat. 200-02) for a week. 4-6×105 PBMCs were then placed in the bottom ofELISPOT plate (nitrocellulose bottomed 96-well plates by MultiScreenMAIP N45; Millipore, cat. MSIPN4W50) pre-coated with IFN-γ capture Ab(Mabtech, cat. 3420-3-1000) and 1-10 μg of arginase derived peptideswere added. PBMCs from each patient and donors were set up in duplicatesor triplicates for peptide and control stimulations. Cells wereincubated in ELISPOT plates in the presence of an antigen for 14-16hours after which they are washed off and secondary biotinylated Ab(Mabtech, cat. 3420-6-1000) was added. After 2 h incubation unboundsecondary antibody was washed off and streptavidin conjugated alkalinephosphatase (AP) (Mabtech, cat. 3310-10) was added for 1 h. Next,unbound conjugated enzyme is washed off and the assay is developed byadding BCIP/NBT substrate (Mabtech, cat. 3650-10). Developed ELISPOTplates were analysed on CTL ImmunoSpot S6 Ultimate-V analyzer usingImmunospot software v5.1. Responses were calculated as the differencebetween average numbers of spots in wells stimulated with Arginase-1peptides and control wells.

HLA-Blocking

For blocking of HLA Class I and II, PBMCs were pre-incubated with 2μg/ml of the blocking antibodies: anti-human HLA-DR, DP, DQ antibodyCloneTü39 (Biolegend, cat. 361702) or anti-human HLA-ABC antibody Clonew6/32 (Dako, Agilent) for 20 min at RT before the addition of thepeptide.

Establishment of Arginase-Specific T-Cell Cultures

Arginase-specific T cell culture were established by stimulation ofcancer patient PBMC with irradiated ArgShort (SEQ ID NO 9)peptide-loaded autologous DC or PBMCs. The following day IL-7 and IL-12(PeproTech, London, UK, cat. 200-07-10 and 200-12) were added.Stimulation of the cultures were carried out every 8 days with ArgShortpeptide loaded irradiated autologous DC followed by ArgShortpeptide-loaded irradiated autologous PBMC. The day after peptidestimulation IL-2 (PeproTech, London, UK, cat. 200-12) was added. After 5stimulations arginase-specific T cells were enriched using TNF-α cellenrichment kit (MiltenyiBiotec, cat. 130-091-269).

Generation of Dendritic Cells

DCs were generated from PBMC by adherence on culture dishes at 37° C.for 1-2 hr. in RPMI-1640. Adherent monocytes were cultured in RPMI-1640supplemented with 10% FCS in the presence of IL-4 (250 U/ml) and GM-CSF(1000 U/ml) (Peprotech, London, UK, cat. 200-04 and 300-03-100) for 6days. DCs were matured by addition of IL-β (1000 U/ml), IL-6 (1000 U/ml)TNF-α (1000 U/ml) (Peprotech, London, UK, cat. 200-01B, 200-06 and300-01A) and PGE2 (1 ug/ml) (Sigma Aldrich, cat. P6532).

B Cell Isolation

PBMCs from cancer patients were thawed and rested overnight. B cellswere isolated from patient PBMCs using Pan B Cell Isolation Kit(Miltenyi Biotec Inc., cat. 130-101-638) according to manufacturer'sinstructions.

Production of In Vitro-Transcribed mRNA

The cDNA encoding Arginase (accession nr. NM_000045) was synthesized andcloned into either pSP73-SphA64 (kindly provided by Dr. E. Gilboa, DukeUniversity Medical Center, Durham, N.C.) using 5′XhoI/3′PacI restrictionsites (Geneart/Life Technologies) or into the HLA class II targetingplasmid pGEM-sig-DC.LAMP (kindly by provided by Dr. K. Thielemans,Medical School of the Vrije Universiteit Brussel) using 5′BamHI/3′BamHIrestriction sites. Both plasmids were linearized with SpeI beforeserving as DNA template for in vitro transcription.

Electroporation

For mRNA experiments, dendritic cells and B cells were transfected withArginase mRNA or control mRNA encoding GFP or nerve growth factorreceptor (NGFR) using electroporation parameters as previouslydescribed. Briefly, cells were washed twice, suspended in Opti-MEMmedium (Invitrogen, cat. 11058021) and adjusted to a final cell densityof 4-7×106 cells/ml. The cell suspension (200-300 ul) was pre-incubatedon ice for 5 min and 5-10 μg of mRNA was added. Cell suspension was thentransferred into a 4-mm gap electroporation cuvette and electroporated.Electroporated cells were further incubated in humidified atmospherewith 5% CO2 and used for experimental analysis as specified.Electroporation efficiency was determined 24 hours later by FACSanalysis of the GFP or NGFR transfected cells.

Flow Cytometric Analysis

Flow cytometry analysis was performed on a FACSCanto™ II (BDBiosciences, San Jose Calif., USA). Intracellular staining of cellcultures was performed after the cells were stimulated with 20-merepeptides for 5 hours or 30-mere peptides for 8 h (BD GolgiPlug™ cat.555029, was added after the first hour). The cells were then stained forsurface markers, then washed and permeabilized by usingFixation/Permeabilization and Permeabilization Buffer (eBioscience, cat.00-5123-43), according to manufacturer's instructions. Antibodies used:IFNγ-APC (cat. 341117), TNFα-BV421 (cat. 562783), CD4-FITC (cat.347413), CD8-PerCP (cat. 345774) (all from BD Biosciences). Dead cellswere stained using FVS510 (564406, BD Biosciences) according tomanufacturer's instructions.

Results

Spontaneous Immune Responses Against Arginase-1

We divided the entire arginase-1 protein sequence into overlapping20-amino-acid-long peptides, generating a library of 31 peptidescovering the whole sequence (SEQ ID NOs 18-48)). Each peptide in thelibrary overlapped with the first 10 amino acids of the followingpeptide. Using this arginase peptide library and the IFNγ ELISPOT assay,we next screened PBMCs from melanoma patients and healthy donors forspontaneous responses (FIGS. 9A and 9B). The PBMCs were stimulated forone week with a pool of 3-4 adjacent 20-mer arginase library peptidesand low-dose IL-2 (120 U/mL). They were then set up for an IFNγ ELISPOTassay to screen for responses against each 20-mer peptide separately.The following eight peptides showed the highest and most abundantresponses in cancer patient PBMCs (peptides labelled by reference tostart and end position): Arg(31-50), Arg(111-130), Arg(161-180),Arg(171-190), Arg(181-200), Arg(191-210), Arg(221-240), andArg(261-280). Among these overlapping peptides, Arg(161-180),Arg(171-190), Arg(181-200), and Arg(191-210) spanned a50-amino-acid-long region that was deemed a hot-spot region since nearlyall patients harbored a response against one or more of these peptides(FIG. 9A). The selected eight peptides were further used to screen forspontaneous immune responses against arginase-1 in PBMCs from eighthealthy donors using IFNγ ELISPOT. As in the PBMCs from cancer patients,the PBMCs from healthy donors showed the highest IFNγ responses againstthe four arginase peptides in the hot-spot region (FIG. 9B).

Arginase-1 Responses in Melanoma TILs

To investigate the potential presence of arginase-specific T cells amongtumor-infiltrating lymphocytes in cancer, we screened TILs from eightmelanoma patients for responses against arginase-1 derived peptides. Tothis end, we performed intracellular staining for IFNγ and TNFα releasein response to peptide stimulation. TILs were thawed and restedovernight without IL-2, and then stimulated for 5 h with three hot-spotimmunogenic region 20-mer peptides (peptides labelled by reference tostart and end position): Arg(161-180), Arg(171-190), Arg(181-200). Inone of the TIL cultures, IFNγ was released from CD4+ T cells in responseto stimulation with all three arginase peptides (FIG. 10A), suggestingthat the arginase-1 hot-spot region likely contained a number of CD4+T-cell epitopes. The percentage of IFNγ-producing cells was higher forArg(171-190) and Arg(181-200) compared to Arg(161-180), and the responseagainst Arg(181-200) was almost twice that against Arg(161-180). No IFNγor TNFα release was observed from CD8+ T cells.

Arginase-1 Hot-Spot Region is Recognized by CD4+ and CD8+ T Cells

Since we most frequently observed responses against arginase-1 peptidesfrom the hot-spot region, we next analyzed whether longer peptidescovering the same sequence could be used instead of four 20-mers. A50-mer peptide covering the entire hot-spot region elicited lowerresponses compared to the 20-mer peptides (data not shown). We thendivided the arginase-1 hot-spot region into two 30-mer peptides thatoverlapped by 10 amino acids (peptides labelled by reference to startand end position): Arg(161-190) and Arg(181-210). These 30-mer peptideswere used to check for responses in selected PBMCs from cancer patientsand healthy donors, which had previously shown responses against the20-mer hot-spot peptides. The PBMCs were stimulated for one week witheither Arg(161-190) or Arg(181-210) in the presence of IL-2, and werethen used in IFNγ ELISPOT. PBMCs from both cancer patients and healthydonors showed high responses against Arg(161-190) (FIG. 10B), comparableto the responses against 20-mer peptides. Some PBMCs also showedresponses against the 30-mer Arg(181-210) peptide (FIG. 10C); however,these responses were lower than those against the overlapping 20-merpeptides covering the same protein region (FIG. 9B).

To investigate which types of T cells reacted to the peptide epitopes inthe hot-spot region, we performed intracellular staining for IFNγ andTNFα release in PBMCs from two healthy donors and one cancer patient,which had shown responses against the Arg(161-190) peptide in IFNγELISPOT. We detected TNFα release from CD4+ T cells after an 8-hourincubation with Arg(161-190) (FIG. 11A). We also detected a minorresponse from CD8+ T cells in some samples (data not shown), suggestingthe presence of HLA Class I and II epitopes in the arginase-1 hot-spotregion. Blocking HLA Class I or Class II expression partially blockedthe IFNγ release in response to Arg(161-190) peptide stimulation,further demonstrating that Arg(161-190) contained CD4+ and CD8+ T cellepitopes (FIG. 11B).

We generated an arginase-specific CD4+ T-cell culture by repeatedstimulation of PBMCs from a melanoma patient with DCs and PBMCs thatwere loaded with a minimal arginase peptide (ArgShort) located in thehot-spot region. The T-cell culture specific against the minimalarginase epitope also recognized the 30-mer Arg(161-190) peptide in IFNγELISPOT (FIG. 11C, left) and intracellular staining (FIG. 11C, right).However, the 50-mer peptide covering the entire hot-spot region was notrecognized. After 8 hours of Arg(161-190) peptide stimulation,intracellular staining revealed TNFα release from CD4+ T cells.

T-Cell Recognition Dependent on Arginase-1 Expression

To assess the ability of arginase-specific CD4+ T cells to recognize andreact against immune cells producing arginase-1, we transfectedautologous dendritic cells and B cells with mRNA encoding arginase-1protein. Autologous DCs were transfected with two different constructsencoding arginase-1 mRNA. One of these constructs contained thearginase-1 sequence fused to the DC-LAMP signal sequence, which targetsa protein towards the lysosomal compartment and thus directs thatprotein towards Class II presentation. Arginase-specific CD4+ T-cellcultures from two different melanoma patients were rested without IL-2for 24 h, and then set up for IFNγ ELISPOT with electroporatedautologous DCs or B cells. We observed higher reactivity against DCs andB cells that were transfected with arginase-1 mRNA compared to Mockcontrol (FIG. 12A-D). The responses were even higher against the DCstransfected with arginase-DC-LAMP compared to both Mock control andarginase-1 mRNA (FIG. 12E). After 24 h, we checked the electroporationefficiency of DCs via FACS analysis of GFP/NGFR-expressing cells,finding >90% transfection efficiency.

CONCLUSION

These experiments confirm that the region spanning positions 161 to 190of Arginasel is particularly immunogenic in both cancer patients andhealthy donors, giving rise to both CD4 and CD8 positive T cellresponses. The region may contain multiple HLA Class I and Class IIrestricted. Peptides derived from this region may be particularlyeffective in a vaccine against Arginasel. Such a vaccine would beexpected to have treatment benefits in cancer.

Example 3—In Vivo Experiments

Design of Peptides for In Vivo Experiments

In order to design peptides suitable for use in vaccination experimentsin mice, the sequence of murine Arginasel (SEQ ID NO: 59) was comparedto the human Arginasel sequence of SEQ ID NO: 1. See alignment shown inFIG. 13, which demonstrates that the sequences have a high level ofsimilarity. The level of similarity is particularly high in the hotspotregion of human Arginasel described in the above examples, i.e. theregion corresponding to positions 161-210 of SEQ ID NO: 1—this 50 aminoacid region is shown in bold for both sequences in FIG. 13. An alignmentof this region and the corresponding region in murine Arginase 1 is alsoshown below:

hArg1: (SEQ ID NO: 52)GFSWVTPCISAKDIVYIGLRDVDPGEHYILKTLGIKYFSMTEVDRLGIGK mArg1:(SEQ ID NO: 57) GFSWVTPCISAKDIVYIGLRDVDPGEHYI I KTLGIKYFSMTEVD K LGIGK

Only 2 residues of the 50 differ, shown as bold and underlined. Aleucine in the human sequence is substituted for the highly similaraliphatic amino acid isoleucine in the mouse, and an arginine in thehuman sequence is substituted for the similarly basic lysine in themouse. Both changes are conservative. Accordingly the hotspot region ishighly conserved between humans and mice.

Given the above similarity, it was determined that relatively fewchanges would be required to create murine analogues of the humanpeptides tested in the previous examples. The peptides used weretherefore as follows:

(SEQ ID NO: 34) GFSWVTPCISAKDIVYIGLR

-   -   ARG17 is the sequence of positions 161-180 of human Arginase 1        (SEQ ID NO: 1). The corresponding region in murine Arginase 1 is        identical. (Also referred to interchangeably as ARG1-17 and        ARG(161-180) in previous Examples).

(SEQ ID NO: 36) DVDPGEHYI L KTLGIKYFSM

-   -   ARG1-19 is the sequence of positions 181-200 human Arginase 1        (SEQ ID NO: 1). The corresponding region in murine Arginasel        differs in a single position (the bold underlined L is replaced        with I in the murine sequence). (Also referred to        interchangeably as ARG(181-200) in previous Examples).

(SEQ ID NO: 53) KDIVYIGL

-   -   mARG1 is the sequence of positions 172-179 of human Arginase 1        (SEQ ID NO: 1). The corresponding region in murine Arginase 1 is        identical.

(SEQ ID NO: 54) LGIKYFSM

-   -   mARG2 is the sequence of positions 193-200 of human Arginase 1        (SEQ ID NO: 1). The corresponding region in murine Arginase 1 is        identical.

(SEQ ID NO: 56) KTLGIKYFSMTEVD K LGIGK

-   -   mARG20 is the sequence of positions 191-210 of murine Arginase 1        (SEQ ID NO: 59). The corresponding region in human Arginasel is        SEQ ID NO: 37 and differs in a single position (the bold        underlined K is replaced with R in the human sequence).        Peptide Vaccination of C57BL/6 and Balb/c Mice

Animals were vaccinated subcutaneously with 100 g peptide in DMSO/H₂O ina 1:1 emulsion with incomplete Freund's adjuvant (IFA) or montanide.IFA+DMSO/H₂O served as a control vaccine. Vaccinations were carried outon day0 and day7. For subsequent analysis of the immune response tospecific peptides, mice were sacrificed on day 14 and spleen anddraining lymph nodes (dLNs) were harvested.

ELISPOT Analysis of Peptide-Specific Response

Murine immune cells were subjected to ELISPOT analysis. Single cellsuspension was prepared from spleen or dLNs by passage through a cellstrainer. After lysis of red blood cells, 0.9×10E6 cells/well wereseeded into ELISPOT plates coated with anti-IFNgamma antibody. Peptideof interest was added to designated wells and cells were incubated o/nwith peptide. The next day, cells were removed, plates washed andincubated with biotinylated detection antibody. Finally, after additionof Streptavidin-ALP and substrate visible spots appear. Each spotcorresponds to an individual IFNgamma producing cell. Plates wereanalysed in an Immunospot analyser and plotted as spots/0.9×10E6 cellsminus background (i.e. minus number of spots in correspondingunstimulated (no peptide) wells).

Tumor Vaccination of Male Balb/c Mice, Age 9-10 Weeks, 4 Animals PerGroup

Each animal was inoculated subcutaneously into the left flank with0.5×10E6 syngeneic CT26.WT colon cancer cells in 100 μl PBS. On day6after inoculation when tumors were palpable, animals received the firstvaccination of peptide or control vaccine (as described above forpeptide vaccination). On day 13, animals received the secondvaccination. Tumor growth was monitored and tumors were measured 3× perweek. Tumor volume was calculated at V [mm³]=1×w²/2 (where 1 is thelongest diameter and w perpendicular to 1).

Results

ARG17, ARG1-19 and mARG20 were all found to be immunogenic in bothC57BL/6 and Balb/c mice. Peptide specific responses were detected inboth C57BL/6 and Balb/c mice vaccinated with each of ARG17, ARG1-19 andmARG20 (see FIGS. 14-15). Of the shorter peptides, mARG1 was immunogenicin C57BL/6 mice and mARG2 was not immunogenic in either strain (data notshown).

In the tumour inoculation experiments, vaccination with each of ARG17,ARG1-19 and mARG20 was found to inhibit tumor growth relative tovaccination with control peptide. The effect was most noticeable withARG1-19 and mARG20, although a treatment benefit was apparent for allthree tested peptides. This confirms the potential of the peptides ofthe invention (and of vaccination against Arginase 1 in general) astreatments for cancer.

The lower responses to ARG17 may reflect a lack of stability of thatpeptide, e.g. due to the presence of a cysteine residue. Replacement ofthat amino acid by conservative substitution could solve this problemwithout altering other properties of the sequence of Arg1 defined bypositions 161-180. However, these results also suggest that the sequenceof Arg1 defined by positions 181-200 and particularly 191-210 may bepreferred in that they are easy to manufacture, stable and appear togenerate best responses in mice.

REFERENCES

-   [1] Bronte V, Zanovello P (2005) Regulation of immune responses by    L-arginine metabolism. Nat Rev Immunol 5:641-54.-   [2] Mussai F, De S C, Abu-Dayyeh I, Booth S, Quek L, McEwen-Smith R    M, et al. (2013) Acute myeloid leukemia creates an    arginase-dependent immunosuppressive microenvironment. Blood    122:749-58.-   [3] Andersen M H, Bonfill J E, Neisig A, Arsequell G, Sondergaard I,    Valencia G, et al. (1999) Phosphorylated Peptides Can Be Transported    by TAP Molecules, Presented by Class I MHC Molecules, and Recognized    by Phosphopeptide-Specific CTL. J Immunol 163:3812-8.-   [4] Ahmad S M, Martinenaite E, Hansen M, Junker N, Borch T H, Met O,    et al. PD-L1 peptide co-stimulation increases immunogenicity of a    dendritic cell-based cancer vaccine. in press ed. 2016.

SEQUENCES Full length human Arginase 1 (NP_000036.2) (SEQ ID NO: 1)MSAKSRTIGI IGAPFSKGQP RGGVEEGPTV LRKAGLLEKL KEQECDVKDY GDLPFADIPNDSPFQIVKNP RSVGKASEQL AGKVAEVKKN GRISLVLGGD HSLAIGSISG HARVHPDLGVIWVDAHTDIN TPLTTTSGNL HGQPVSFLLK ELKGKIPDVP  GFSWVTPCIS AKDIVYIGLRDVDPGEHYIL KTLGIKYFSM TEVDRLGIGK  VMEETLSYLL GRKKRPIHLS FDVDGLDPSFTPATGTPVVG GLTYREGLYI TEEIYKTGLL SGLDIMEVNP SLGKTPEEVT RTVNTAVAITLACFGLAREG NHKPIDYLNP PKRegion identified as a hotspot for immunogenicity shown bold and underlinedNOTE: the above sequence is isoform2 of human Arginase 1, which has the mostwidespread distribution of expression. An alternative, longer isoforml is expressedpredominantly in the liver. The sequence for this form is provide as NP_001231367.1.It includes an additional 8 amino acid sequence (VTQNFLIL, SEQ ID NO: 62) insertedbetween positions corresponding to positions 43 and 44 of isoform 2 as shown in SEQID NO: 1. Any polypeptide of the invention which spans those positions of isoform2may optionally include the insertion of the said additional 8 amino acids of isoform1.Full length murine Arginase 1 (NP_031508.1) (SEQ ID NO: 59)MSSKPKSLEI IGAPFSKGQP RGGVEKGPAA LRKAGLLEKL KETEYDVRDH GDLAFVDVPNDSSFQIVKNP RSVGKANEEL AGVVAEVQKN GRVSVVLGGD HSLAVGSISG HARVHPDLCVIWVDAHTDIN TPLTTSSGNL HGQPVSFLLK ELKGKFPDVP  GFSWVTPCIS AKDIVYIGLRDVDPGEHYII KTLGIKYFSM TEVDKLGIGK  VMEETFSYLL GRKKRPIHLS FDVDGLDPAFTPATGTPVLG GLSYREGLYI TEEIYKTGLL SGLDIMEVNP TLGKTAEEVK STVNTAVALTLACFGTQREG NHKPGTDYLK PPKRegion identified as a hotspot for immunogenicity shown bold and underlinedFull length human Arginase 2 (NP_001163.1) (SEQ ID NO: 60)MSLRGSLSRL LQTRVHSILK KSVHSVAVIG APFSQGQKRK GVEHGPAAIR EAGLMKRLSSLGCHLKDFGD LSFTPVPKDD LYNNLIVNPR SVGLANQELA EVVSRAVSDG YSCVTLGGDHSLAIGTISGH ARHCPDLCVV WVDAHADINT PLTTSSGNLH GQPVSFLLRE LQDKVPQLP GFSWIKPCISS ASIVYIGLRD VDPPEHFILK NYDIQYFSMR DIDRLGIQK V MERTFDLLIGKRQRPIHLSF DIDAFDPTLA PATGTPVVGG LTYREGMYIA EEIHNTGLLS ALDLVEVNPQLATSEEEAKT TANLAVDVIA SSFGQTREGG HIVYDQLPTP SSPDESENQA RVRIRegion identified as a hotspot for immunogenicity shown bold and underlined

TABLE A SEQ Start End ID NO Sequence Name pos pos  2 QLAGKVAEV Arg1  79 87  3 LVLGGDHSL Arg2  95 103  4 LLKELKGKI Arg3 148 156  5 ELKGKIPDVArg4 151 159  6 VMEETLSYL Arg5 211 219  7 HLSFDVDGL Arg6 228 236  8SLGKTPEEV Arg7 281 289  9 IVYIGLRDV Arg8 174 182 10 GLLSGLDIM Arg9 268276 11 DIMEVNPSL Arg10 274 282 12 LLSGLDIMEV Arg11 269 278 13 NLHGQPVSFLArg12 139 148 14 SLVLGGDHSL Arg13  94 103 15 YLLGRKKRPI Arg14 218 227 16VLGGDHSLAI Arg15  96 105 17 FLLKELKGKI Arg16 147 156 18MSAKSRTIGIIGAPFSKGQP Arg1-1   1  20 19 IGAPFSKGQPRGGVEEGPTV Arg1-2  11 30 20 RGGVEEGPTVLRKAGLLEKL Arg1-3  21  40 21 LRKAGLLEKLKEQECDVKDYArg1-4  31  50 22 KEQECDVKDYGDLPFADIPN Arg1-5  41  60 23GDLPFADIPNDSPFQIVKNP Arg1-6  51  70 24 DSPFQIVKNPRSVGKASEQL Arg1-7  61 80 25 RSVGKASEQLAGKVAEVKKN Arg1-8  71  90 26 AGKVAEVKKNGRISLVLGGDArg1-9  81 100 27 GRISLVLGGDHSLAIGSISG Arg1-10  91 110 28HSLAIGSISGHARVHPDLGV Arg1-11 101 120 29 HARVHPDLGVIWVDAHTDIN Arg1-12 111130 30 IWVDAHTDINTPLTTTSGNL Arg1-13 121 140 31 TPLTTTSGNLHGQPVSFLLKArg1-14 131 150 32 HGQPVSFLLKELKGKIPDVP Arg1-15 141 160 33ELKGKIPDVPGFSWVTPCIS Arg1-16 151 170 34 GFSWVTPCISAKDIVYIGLR Arg1-17 161180 35 AKDIVYIGLRDVDPGEHYIL Arg1-18 171 190 36 DVDPGEHYILKTLGIKYFSMArg1-19 181 200 37 KTLGIKYFSMTEVDRLGIGK Arg1-20 191 210 38TEVDRLGIGKVMEETLSYLL Arg1-21 201 220 39 VMEETLSYLLGRKKRPIHLS Arg1-22 211230 40 GRKKRPIHLSFDVDGLDPSF Arg1-23 221 240 41 FDVDGLDPSFTPATGTPVVGArg1-24 231 250 42 TPATGTPVVGGLTYREGLYI Arg1-25 241 260 43GLTYREGLYITEEIYKTGLL Arg1-26 251 270 44 TEEIYKTGLLSGLDIMEVNP Arg1-27 261280 45 SGLDIMEVNPSLGKTPEEVT Arg1-28 271 290 46 SLGKTPEEVTRTVNTAVAITArg1-29 281 300 47 RTVNTAVAITLACFGLAREG Arg1-30 291 310 48LACFGLAREGNHKPIDYLNPPK Arg1-31 301 322 49 GLYITEEIYKTGLLSGLDIM 257 27650 GFSWVTPCISAKDIVYIGLRDVDPGEHYIL 161 190 51DVDPGEHYILKTLGIKYFSMTEVDRLGIGK 181 210 52GFSWVTPCISAKDIVYIGLRDVDPGEHYILKT 161 210 LGIKYFSMTEVDRLGIGK 53 KDIVYIGLmARG1 172 179 54 LGIKYFSM mARG2 193 200 55 DVDPGEHYI I KTLGIKYFSM* 181200 56 KTLGIKYFSMTEVD K LGIGK* mARG20 191 210 57GFSWVTPCISAKDIVYIGLRDVDPGEHYI I KT 161 210 LGIKYFSMTEVD K LGIGK* 58GFSWIKPCISSASIVYIGLRDVDPPEHFILKN YDIQYFSMRDIDRLGIQK^(#)*indicates a sequence from murine Arginase 1 which includes at least one difference relative to the corresponding region of humanArginase1. Residues which are non-identical with the correspondinghuman sequence are bold and underlined. Murine and human Arginase 1are the same length so start and end positions are the same. ^(#)indicates a sequence from human Arginase 2. Start and end positionsare the corresponding positions in human Arginase 1. Alternative human Arginase 1 sequence (SEQ ID NO: 61)MSAKSRTIGIIGAPFSKGQPRGGVEEGPTVLRAGLLEKLKEQECDVKDYGDLPFADIPNDSPFQIVKNPRSVGKASEQLAGKVAEVKKNGRISLVLGGDHSLAISIGHARVHPDLGVIWVDAHTDINTPLTTTSGNLHGQPVSFLLKELKGKIPDVPGFSWVTPCISAKDIVYIGLRDVDPGEHYILKTLGIKYFSMTEVDRLGIGKVMEETLSYLLGRKRPIHLSFDVDGLDPSFTPATGTPVVGGLTYREGLYITEEIYKTGLLSGLDIMEVNPSLGKTPEEVTRTVNTAVAITLACFGLAREGNHKPIDYLNPPK

The invention claimed is:
 1. An isolated, immunogenic polypeptidefragment of a human Arginase protein of SEQ ID NO: 1 (Arginase 1) or SEQID NO: 60 (Arginase 2), wherein (a) the fragment of a human Arginase 1protein is up to 55 amino acids in length and comprises the amino acidsequence of SEQ ID NO: 36 or SEQ ID NO: 53, or (b) the fragment of ahuman Arginase 2 protein is 8 to 55 amino acids in length and comprisesat least 8 consecutive amino acids of SEQ ID NO:
 58. 2. The polypeptidefragment of claim 1, which is a fragment of a human Arginase 1 proteinand comprises the amino acid sequence of any one of SEQ ID NOs: 4-6, 9,13, 15, 17, 31-39 or 50-54.
 3. The polypeptide fragment of claim 1,which comprises at least 8, 9, 10, 15 or all 20 consecutive amino acidsof any one of SEQ ID NOs: 37, 36, 34 or
 35. 4. The polypeptide fragmentof claim 1, wherein: a. the C terminal amino acid is replaced with thecorresponding amide; and/or b. the fragment comprises an amino acidcorresponding to position 190 of SEQ ID NO: 1 and the L at the positioncorresponding to position 190 of SEQ ID NO: 1 is replaced with I; and/orc. the fragment comprises an amino acid corresponding to position 205 ofSEQ ID NO: 1 and the R at the position corresponding to position 205 ofSEQ ID NO: 1 is replaced with K; and/or d. at least one additionalmoiety is attached to the N and/or C terminus of the fragment to improvesolubility or manufacturability of the polypeptide fragment; and/or e.said fragment lacks or has reduced arginase activity relative to thecorresponding full length arginase; and/or f. said fragment is capableof stimulating T cells which recognize cells expressing thecorresponding arginase.
 5. A composition comprising the polypeptidefragment of claim 1 and a pharmaceutically acceptable diluent orcarrier.
 6. The composition of claim 5, which further comprises anadjuvant selected from the group consisting of bacterial DNA basedadjuvants, oil/surfactant based adjuvants, viral dsRNA based adjuvants,imidazochinilines, and a Montanide ISA adjuvant.
 7. A method of treatingor preventing a disease or condition in a subject, the method comprisingadministering to the subject an isolated, immunogenic polypeptidefragment of a human Arginase protein of SEQ ID NO: 1 (Arginase 1) or SEQID NO: 60 (Arginase 2), or a composition comprising the isolatedpolypeptide fragment and a pharmaceutically acceptable diluent orcarrier, wherein: (a) the fragment of a human Arginase 1 protein is upto 55 amino acids in length and comprises the amino acid sequence of SEQID NO: 36 or SEQ ID NO: 53, or (b) the fragment of a human Arginase 2protein is 8 to 55 amino acids in length and comprises at least 8consecutive amino acids of SEQ ID NO:
 58. 8. The method of claim 7,wherein the disease or condition is characterized at least in part byinappropriate or excessive immune suppressive function of an Arginase,and/or said disease or condition is cancer.
 9. The method of claim 8,wherein the disease or condition is cancer and the method furthercomprises the simultaneous or sequential administration of an additionalcancer therapy.
 10. The method of claim 9, wherein the additional cancertherapy is one or more of Actimide, Azacitidine, Azathioprine,Bleomycin, Carboplatin, Capecitabine, Cisplatin, Chlorambucil,Cyclophosphamide, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine,Doxorubicin, Epirubicin, Etoposide, Fludarabine, Fluorouracil,Gemcitabine, Hydroxyurea, Idarubicin, Irinotecan, Lenalidomide,Leucovorin, Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate,Mitoxantrone, Nivolumab, Oxaliplatin, Paclitaxel, Pembrolizumab,Pemetrexed, Revlimid, Temozolomide, Teniposide, Thioguanine, Valrubicin,Vinblastine, Vincristine, Vindesine and Vinorelbine.
 11. The method ofclaim 7, wherein the polypeptide fragment is a fragment of a humanArginase 1 protein and comprises the amino acid sequence of any one ofSEQ ID NOs: 4-6, 9, 13, 15, 17, 31-39 or 50-54.
 12. The method of claim7, wherein the polypeptide fragment comprises at least 8, 9, 10, 15 orall 20 consecutive amino acids of any one of SEQ ID NOs: 37, 36, 34 or35.
 13. The method of claim 7, wherein: a. the C terminal amino acid ofthe polypeptide fragment is replaced with the corresponding amide;and/or b. the fragment comprises an amino acid corresponding to position190 of SEQ ID NO: 1 and the L at the position corresponding to position190 of SEQ ID NO: 1 of the polypeptide fragment is replaced with I;and/or c. the fragment comprises an amino acid corresponding to position205 of SEQ ID NO: 1 and the R at the position corresponding to position205 of SEQ ID NO: 1 of the polypeptide fragment is replaced with K;and/or d. at least one additional moiety is attached to the N and/or Cterminus of the polypeptide fragment to improve solubility ormanufacturability of the polypeptide fragment; and/or e. the polypeptidefragment lacks or has reduced arginase activity relative to thecorresponding full length arginase; and/or f. the polypeptide fragmentis capable of stimulating T cells which recognize cells expressing thecorresponding arginase.
 14. The method of claim 7, which comprisesadministering to the subject a composition comprising said polypeptidefragment and an adjuvant selected from the group consisting of bacterialDNA based adjuvants, oil/surfactant based adjuvants, viral dsRNA basedadjuvants, imidazochinilines, and a Montanide ISA adjuvant.
 15. Anucleic acid sequence encoding the immunogenic polypeptide fragment ofclaim
 1. 16. A vector comprising the nucleic acid sequence of claim 15.17. A method of treating or preventing a disease or condition in asubject, which method comprises administering to a subject in needthereof a composition comprising the nucleic acid sequence of claim 15and a pharmaceutically acceptable carrier.
 18. The method of claim 17,wherein the disease or condition is characterized by inappropriate orexcessive immune suppressive function of an Arginase, and/or the diseaseor condition is cancer.
 19. A method of treating or preventing a diseaseor condition in a subject, which method comprises administering to asubject in need thereof a composition comprising the vector of claim 16and a pharmaceutically acceptable carrier.
 20. The method of claim 19,wherein the disease or condition is characterized by inappropriate orexcessive immune suppressive function of an Arginase, and/or the diseaseor condition is cancer.
 21. The polypeptide fragment of claim 3, whichcomprises SEQ ID NO: 34 except that the cysteine in SEQ ID NO: 34 isreplaced by a conservative amino acid substitution.
 22. The polypeptidefragment of claim 4, wherein at least one additional moiety is attachedto the N and/or C terminus to improve solubility or manufacturability ofthe polypeptide fragment, and wherein said additional moiety is ahydrophilic amino acid.
 23. The polypeptide fragment of claim 22,wherein said hydrophilic amino acid is R or K.
 24. The composition ofclaim 5, further comprising an adjuvant.
 25. The method according toclaim 9, wherein the additional cancer therapy is a cytokine therapy, aT-cell therapy, an NK therapy, an immune system checkpoint inhibitor,chemotherapy, radiotherapy, immunostimulating substances, gene therapy,or dendritic cells.
 26. The method according to claim 25, wherein theimmune system checkpoint inhibitor is an antibody.
 27. The method ofclaim 12, wherein the polypeptide fragment comprises SEQ ID NO: 34except that the cysteine in SEQ ID NO: 34 is replaced by a conservativeamino acid substitution.
 28. The method of claim 13, wherein at leastone additional moiety is attached to the N and/or C terminus of thepolypeptide fragment to improve solubility or manufacturability of thepolypeptide fragment, and wherein said additional moiety is ahydrophilic amino acid.
 29. The method of claim 28, wherein saidhydrophilic amino acid is R or K.